Selective inhibitors of Alpha2-containing isoforms of Na,K-ATPase and use thereof for reduction of intraocular pressure

ABSTRACT

Provided herein are alpha2-selective Na,K-ATPase inhibitors and prodrugs thereof, characterized by having a cyclic moiety attached to a digoxin or digitoxin derivative, as well as uses thereof in lowering intraocular pressure and in treating glaucoma and heart conditions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/745,441 filed on Jan. 17, 2018, which is a National Phase of PCTPatent Application No. PCT/IL2016/050785 having International FilingDate of Jul. 19, 2016, which claims the benefit of priority under 35 USC§ 119(e) of U.S. Provisional Patent Application No. 62/302,226, filed onMar. 2, 2016. PCT Patent Application No. PCT/IL2016/050785 is also aContinuation-in-Part (CIP) of PCT Patent Application No.PCT/IL2015/050741, having international Filing Date of Jul. 19, 2015.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 82372SequenceListing.txt, created on Apr. 13,2020, comprising 73,501 bytes, submitted concurrently with the filing ofthis application is incorporated herein by reference. The sequencelisting submitted herewith is identical to the sequence listing formingpart of the international application.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates topharmaceutical agents and, more particularly, but not exclusively, todigoxin and digitoxin derivatives exhibiting selective inhibition ofα2-containing isoforms of Na,K-ATPase, and uses thereof to reduceintraocular pressure (IOP), and/or as cardiotonic agents in a subject inneed thereof.

Glaucoma is a disease leading to irreversible blindness. Control ofintraocular pressure (IOP) is the mainstay of current therapy ofglaucoma, and is achieved by various drugs, such as β-blockers,prostaglandin analogues, α2 adrenergic receptor agonists, cholinergicagonists and carbonic anhydrase inhibitors given topically orsystemically. The topical route is preferable, provided the drugeffectively permeates the cornea, because this minimizes systemicside-effects. Despite the selection of drugs available, uncontrolled IOPin many patients eventually makes surgical intervention necessary. Thus,fresh approaches to drug treatment of glaucoma are highly desirable.

The Na,K-ATPase is the motor for production of the aqueous humour(bodily fluid) in the ciliary body epithelium and, in principle,inhibition of the Na,K-ATPase can suppress the production of aqueoushumour, and control IOP. Control of IOP is the mainstay of glaucomatherapy; however, the available drugs suffer from a variety ofshortcomings, particularly due to systemic adverse effects and lowtherapeutic index. Previously, intra-venous digoxin, a classicalinhibitor of the Na,K-pump, typically used primarily to treat congestiveheart failure, was considered for this role but was discarded due tosystemic toxicity.

Isoforms of the Na,K-ATPase ion pump consists of α and β subunits (α/β)and accessory FXYD regulatory subunits. There are four isoforms of theα1 subunit (α1-4) and three isoforms of the β subunit (β1-3) expressedin a tissue-specific fashion. The α1 isoform is the common isoform thatmaintains Na and K gradients in all tissues, α2 is expressed mainly inmuscle and astrocytes, and α3 is expressed mainly in nerve cells. Forexample, human heart expresses α1 (about 70%) and both α2 and α3isoforms (about 30%) and β1.

The ciliary epithelium in the eye is a functional syncytium consistingof apical pigmented cells (PE) oriented towards the blood andbaso-lateral non-pigmented (NPE) cells oriented towards the anteriorchamber of the eye. It is known that the primary Na,K-ATPase isoform ofthe PE is α1β1 while that of the NPE is α2β3. The Na,K-ATPase in the NPEpowers the production of the aqueous humor and controls intraocularpressure.

Thus, in principle, topically applied α2-selective cardiac glycosidesthat penetrate the intact eye and reach the ciliary epithelium couldeffectively reduce IOP. A potential advantage of topical applicationcould be that systemic toxic effects typical of cardiac glycosidesshould be minimal.

Another possible application of an α2-selective cardiac glycoside couldbe as an effective cardiotonic drug, with reduced cardiotoxicity,compared to known drugs such as digoxin. Digitalis drugs such as digoxinhave been used to treat heart failure for over two hundred years but aredangerous drugs with multiple side effects. There is now good evidencethat selective inhibition of α2 is especially effective in enhancingcardiac excitation-contraction coupling and mediating cardiacglycoside-mediated positive inotropy. Inhibition of α2, which is a minorisoform and is located largely in T-tubules, may mediate the positivecardiotonic effects, but α2-selective cardiac glycosides should onlyminimally inhibit α1, located primarily in the outer sarcolemmamembrane, and thus avoid cellular Ca overload, the hallmark of cardiactoxicity.

The isoform selectivity of a large number of known cardiac glycosideshas been previously studied, using the yeast P. pastoris expressingNa,K-ATPase isoforms (α1β1, α2β1, β3β1), and purified detergent-solubleisoform complexes of Na,K-ATPase [Cohen E. et al., 2005, J Biol Chem,280(17), pp. 16610-16618; Haviv H, et al., 2007, Biochemistry, 46(44),pp. 12855-12867; Lifshitz Y, et al., 2007, Biochemistry, 46(51), pp.14937-14950; Mishra N K, et al., 2011, J Biol Chem, 286(11), pp.9699-9712; and Kapri-Pardes E, et al., 2011, J Biol Chem, 286(50), pp.42888-42899].

Dissociation constants, K_(D), for digitalis glycosides, digoxin anddigitoxin, measured by ³H-ouabain displacement assays in membranes,showed moderate selectivity (3-4-fold) for α2/α3 over α1. By contrast tothe digitalis glycosides, the K_(D) of ouabain showed some preferencefor α1 over α2 and similar Ki values for all three isoforms. In assaysof inhibition of Na,K-ATPase activity, measured with the purifiedisoform protein complexes, digoxin and digitoxin showed 3-4-fold lowerKi (inhibition) values for α2 compared to α1, with α3 more similar toα1. No aglycones of any cardiac glycosides tested showed isoformselectivity. For digoxin derivatives, with one to four digitoxosemoieties the maximal α2/α1 selectivity was found for digoxin itself,with three digitoxose sugars [Katz, A. et al., J Biol Chem, 2010,285(25), pp. 19582-19592].

Based on recent studies [Laursen, M. et al., Proc Natl Acad Sci USA,2015, 112(6):1755-60], it was inferred that the sugar moiety of digoxinlikely determines isoform selectivity, which is generally consistentwith recent structures of Na,K-ATPase with bound ouabain, bufalin ordigoxin. The unsaturated lactone ring and steroid portion of ouabain arebound between trans-membrane segments M1, M4, M5 of the c subunit, inwhich there are no amino-acid differences between isoforms. Assumingthat the aglycones of all cardiac glycosides bind similarly, theimplication is that isoforms cannot discriminate between any of theaglycones, as found experimentally. By contrast, the sugar is bound nearextracellular loops, where there are a number of amino-acid differencesbetween the isoforms. These residues might interact with the sugars ofbound digoxin in an isoform-selective way.

Additional background art include WO 2015/029035, WO 2007/079128, WO2010/053771, U.S. Patent Application No. 2005/0032138 and U.S. Pat. Nos.7,888,059 and 7,851,145; these documents are hereby incorporated byreference.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a compound represented by general Formula I:

including any pharmaceutically acceptable salt, prodrug, hydrate,solvate, enantiomer and diastereomer thereof, and any mixtures thereof,

wherein:

X is H or OH;

R is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety;

or

B is selected from the group consisting of an alkylsulfonyl, anarylsulfonyl and a sulfonamide;

or

B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkylprovided that at least one of R₁ and R₂ is a C₁-C₄ alkyl.

According to some embodiments of the invention, A is selected from thegroup consisting of a covalent bond, an unsubstituted C₁-C₆ alkyl, asubstituted C₁-C₆ alkyl, an unsubstituted C₁-C₆ alkyl interrupted by oneor more heteroatom and a substituted C₁-C₆ alkyl interrupted by one ormore heteroatom.

According to some embodiments of the invention, B is a cyclic moietyselected from the group consisting of an unsubstituted alicyclic moiety,a substituted alicyclic moiety, an unsubstituted heterocyclic moiety, asubstituted heterocyclic moiety, an unsubstituted aryl moiety, asubstituted aryl moiety, an unsubstituted heteroaryl moiety and asubstituted heteroaryl moiety.

According to some embodiments of the invention, B is an unsubstitutedalicyclic moiety.

According to some embodiments of the invention, the unsubstitutedalicyclic moiety is selected from the group consisting of cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

According to some embodiments of the invention, B is a substitutedalicyclic moiety.

According to some embodiments of the invention, the substitutedalicyclic moiety is selected from the group consisting of2,3-dimethylcyclopropane-1-yl, 3,3-dimethylcyclobutane-1-yl,3,4-dimethylcyclopentane-1-yl and 3,5-dimethylcyclohexane-1-yl.

According to some embodiments of the invention, B is an unsubstitutedheterocyclic moiety.

According to some embodiments of the invention, the unsubstitutedheterocyclic moiety is selected from the group consisting of oxiranyl,aziridinyl, oxetanyl, azetidinyl, thietanyl, tetrahydrofuranyl,pyrrolidinyl, tetrahydropyranyl and piperidinyl.

According to some embodiments of the invention, B is an unsubstitutedaryl moiety.

According to some embodiments of the invention, the unsubstituted arylmoiety is selected from the group consisting of cyclopentadienyl, phenyland naphthyl.

According to some embodiments of the invention, B is an unsubstitutedheteroaryl moiety.

According to some embodiments of the invention, the unsubstitutedheteroaryl moiety is imidazolyl.

According to some embodiments of the invention, the alkylsulfonyl isselected from the group consisting of methylsulfonyl, ethylsulfonyl andisopropylsulfonyl.

According to some embodiments of the invention, the arylsulfonyl isselected from the group consisting of phenylsulfonyl, benzylsulfonyl andtosyl.

According to some embodiments of the invention, the sulfonamide isselected from the group consisting of methylsulfonamide,N-methylmethanesulfonamide and N,N-dimethylmethanesulfonamide.

According to some embodiments of the invention, B is —N(Et)₂.

According to some embodiments of the invention, X is H.

According to some embodiments of the invention, A is a covalent bond andB is cyclobutyl.

According to some embodiments of the invention, A is —CH₂— and B iscyclopropyl.

According to some embodiments of the invention, X is OH.

According to some embodiments of the invention, A is a covalent bond andB is selected from the group consisting of cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl.

According to some embodiments of the invention, A is —CH₂— and B isselected from the group consisting of cyclopropyl,3,3-dimethylcyclobutane-1-yl and phenyl.

According to some embodiments of the invention, A is —(CH₂)₂— and B iscyclopropyl.

According to some embodiments of the invention, R is selected from thegroup consisting of cyclopropyl, methylcyclopropane, ethylcyclopropane,propylcyclopropane, cyclobutyl, methylcyclobutane,methyl-3,3-dimethylcyclobutane, ethylcyclobutane, propylcyclobutane,cyclopentyl, methylcyclopentane, ethylcyclopentane, propylcyclopentane,cyclohexyl, azetidinyl, oxetanyl, thietanyl, histaminyl and benzyl.

According to some embodiments of the invention, R is selected from thegroup consisting of cyclopropyl, methylcyclopropane and cyclobutyl.

According to some of any of the embodiments of the invention, thecompound is having an affinity to at least one isoform of Na,K-ATPase.

According to some embodiments of the invention, the isoform is selectedfrom the group consisting of α1β1, α1β2, α1β3, α2β1, β2β2, β2β3, α3β1,α3β2, α3β3, α4β1, α4β2 and α4β3.

According to some embodiments of the invention, the affinity of thecompound to any one of α2β1, α2β2 and α2β3 is higher than the affinityto α1β1, α1δ2, α1β3, α3β1, α3β2, α3β3, α4β1, α4β2 and α4β3 by at least100%.

According to some embodiments of the invention, the affinity of thecompound to any one of α2β1, α2β2 and α2β3 is higher than the affinityto α1β1 by at least 300% (4-fold).

According to some embodiments of the invention, the affinity of thecompound to α2β3 is higher than the affinity to α1β1 by at least 500%(6-fold).

According to some embodiments of the invention, the compound is having aα2β3 inhibition constant (Ki) lower than 10 nM.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition that includes as anactive ingredient a compound according to any of the embodiments of theinvention and a pharmaceutically acceptable carrier.

According to some embodiments of the invention, the pharmaceuticalcomposition is packaged in a packaging material and identified in print,or on the packaging material, for use in reducing intraocular pressure(IOP).

According to some embodiments of the invention, the pharmaceuticalcomposition is packaged in a packaging material and identified in print,or on the packaging material, for use in a treatment of a heartcondition.

According to an aspect of some embodiments of the present inventionthere is provided a method of reducing intraocular pressure (IOP) in asubject in need thereof, which includes administering to the subject atherapeutically effective amount of a compound according to any of theembodiments of the invention, or a pharmaceutical composition oaccording to some of the embodiments of the invention.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a heart condition in a subject inneed thereof, that includes administering to the subject atherapeutically effective amount of a compound according to any of theembodiments of the invention, or a pharmaceutical composition accordingto some of the embodiments of the invention.

According to an aspect of some embodiments of the present inventionthere is provided a use of a compound according to any of theembodiments of the invention, or a pharmaceutical composition accordingto some of the embodiments of the invention, for the manufacture of amedicament for reducing intraocular pressure (IOP).

According to an aspect of some embodiments of the present inventionthere is provided a use of a compound according to any of theembodiments of the invention, or a pharmaceutical composition accordingto some of the embodiments of the invention, for the manufacture of amedicament for treating a heart condition.

According to some embodiments of the invention, the heart condition isselected from the group consisting of atrial fibrillation, atrialflutter, mitral stenosis, chronic heart failure and congestive heartfailure.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition that includes as activeingredients:

at least one ingredient selected from the group consisting of aprostaglandin analog, a β-blocker, an adrenergic agent, an α2-adrenergicreceptor agonist, a miotic agent, a carbonic anhydrase inhibitor and acholinergic agonist; and

a compound represented by Formula III:

including any pharmaceutically acceptable salt, prodrug, hydrate,solvate, enantiomer and diastereomer thereof, and any mixtures thereof,and a pharmaceutically acceptable carrier,

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆haloalkyl (C₁-C₆ alkyl substituted with at least one halo),—(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂,—(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R1, R2 and R3 are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl anda C₁-C₄ hydroxyalkyl;

Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or R′ is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of analkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂,wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl providedthat at least one of R₁ and R₂ is a C₁-C₄ alkyl,

and a pharmaceutically acceptable carrier.

According to some embodiments of the invention, the pharmaceuticalcomposition is packaged in a packaging material and identified in print,or on the packaging material, for use in reducing intraocular pressure(IOP).

According to an aspect of some embodiments of the present inventionthere is provided a use of an agent selected from the group consistingof a prostaglandin analog, a β-blocker, an adrenergic agent, anα2-adrenergic receptor agonist, a miotic agent, a carbonic anhydraseinhibitor and a cholinergic agonist, and a compound represented byFormula III:

including any pharmaceutically acceptable salt, prodrug, hydrate,solvate, enantiomer and diastereomer thereof, and any mixtures thereof,

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆haloalkyl (C₁-C₆ alkyl substituted with at least one halo),—(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂,—(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R1, R2 and R3 are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl anda C₁-C₄ hydroxyalkyl;

Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or R′ is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of analkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂,wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl providedthat at least one of R₁ and R₂ is a C₁-C₄ alkyl,

for the manufacture of a medicament for reducing intraocular pressure(IOP).

According to an aspect of some embodiments of the present inventionthere is provided a method of reducing intraocular pressure (IOP) in asubject in need thereof, which includes co-administering to the subjecta therapeutically effective amount of:

an agent selected from the group consisting of a prostaglandin analog, aβ-blocker, an adrenergic agent, an α2-adrenergic receptor agonist, amiotic agent, a carbonic anhydrase inhibitor and a cholinergic agonist;and

a compound represented by Formula III:

including any pharmaceutically acceptable salt, prodrug, hydrate,solvate, enantiomer and diastereomer thereof, and any mixtures thereof,

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆haloalkyl (C₁-C₆ alkyl substituted with at least one halo),—(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂,—(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R₁, R₂ and R₃ are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl anda C₁-C₄ hydroxyalkyl;

Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or R′ is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of analkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂,wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl providedthat at least one of R₁ and R₂ is a C₁-C₄ alkyl.

According to some embodiments of the invention, the mode ofadministration is effected topically, extraocularly, intraocularlyand/or intravitreally.

According to some embodiments of the invention, the compositionaccording to some embodiments of the invention is formulated as anophthalmic composition suitable for topical, extraocular, intraocularand/or intravitreal administration to the eye of the subject.

According to some embodiments of the invention, the compositionaccording to some embodiments of the invention is in the form selectedfrom the group consisting of an eye-drop solution, a spray, an eye washsolution, an ointment, a suspension, a gel, a cream and an injectablesolution.

According to some embodiments of the invention, the intraocular pressure(IOP) is associated with glaucoma, low-tension glaucoma andnormal-tension glaucoma.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a heart condition in a subject inneed thereof, that includes co-administering to the subject atherapeutically effective amount of:

an agent selected from the group consisting of a β-blocker, ananticoagulation agent, an angiotensin-converting-enzyme inhibitor and anangiotensin II receptor antagonist; and

a compound represented by Formula III:

including any pharmaceutically acceptable salt, prodrug, hydrate,solvate, enantiomer and diastereomer thereof, and any mixtures thereof,

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆haloalkyl (C₁-C₆ alkyl substituted with at least one halo),—(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂,—(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R₁, R₂ and R₃ are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

Rb, Rc and Rd are each independently selected from H, a C₁-C₄ alkyl anda C₁-C₄ hydroxyalkyl;

Re is selected from a C₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or R′ is represented by general Formula II:

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of analkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂,wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl providedthat at least one of R₁ and R₂ is a C₁-C₄ alkyl.

According to an aspect of some embodiments of the present inventionthere is provided a process of preparing a compound according any of theembodiments of the invention, the process includes:

converting the third digitoxose moiety of digoxin or digitoxin into adialdehyde; and

reacting the dialdehyde with a reagent represented by general formulaIV:B-A-NH₂   Formula IV

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety, or B is selected from the group consisting of analkylsulfonyl, an arylsulfonyl and a sulfonamide, or B is —NR₁R₂,wherein R₁ and R₂ are each independently H or a C₁-C₄ alkyl providedthat at least one of R₁ and R₂ is a C₁-C₄ alkyl.

According to some embodiments of the invention, converting the thirddigitoxose moiety of digoxin or digitoxin into a dialdehyde is effectedby sodium periodate (NaIO₄).

According to some embodiments of the invention, reacting the dialdehydewith a reagent represented by general formula IV is effected in thepresence of NaCNBH₃.

According to an aspect of some embodiments of the present inventionthere is provided a method of determining an affinity of a compound,according to any embodiment of the invention, to at least one isoform ofNa,K-ATPase, the method includes contacting the isoform of Na,K-ATPasewith the compound in an affinity measurement setup and determining theaffinity.

According to an aspect of some embodiments of the present inventionthere is provided a method of isolating an isoform of Na,K-ATPase of amammal, that includes:

transforming yeast cells with a clone that that includes an α chainsequence and a β chain sequence of the Na,K-ATPase;

expressing the clone in the yeast cells; and

isolating the isoform,

wherein:

the α chain sequence is selected from the group consisting of α1, α2, α3and α4; and

the β chain sequence is selected from the group consisting of β1, β2 andβ3.

According to some embodiments of the invention, the isoform is selectedfrom the group consisting of α1β1, β1β2, α1β3, α2β1, α2β2, α2β3, α3β1,α3β2, α3β3, α4β1, α4β2 and α4β3.

According to some embodiments of the invention, the isoform is α2β2.

According to some embodiments of the invention, the isoform is α2β3.

According to an aspect of some embodiments of the present inventionthere is provided an isolated isoform of Na,K-ATPase of a mammal havingat least 70% purity, wherein the isoform is α2β2.

According to an aspect of some embodiments of the present inventionthere is provided an isolated isoform of Na,K-ATPase of a mammal havingat least 70% purity, wherein the isoform is α2β3.

According to an aspect of some embodiments of the present inventionthere is provided an isolated isoform of Na,K-ATPase of a mammal havinga yeast-characterizing glycosylation pattern, wherein the isoform isα2β2.

According to an aspect of some embodiments of the present inventionthere is provided an isolated isoform of Na,K-ATPase of a mammal havinga yeast-characterizing glycosylation pattern, wherein the isoform isα2β3.

According to some embodiments of the invention, the isolated isoform,according to any embodiment of the invention, is human.

According to some embodiments of the invention a prodrug of the compoundis represented by Formula V:

wherein X is H or PD₃, and

each of PD₁-PD₄ is H or independently selected from the group consistingof a methoxymethyl ether, a tetrahydropyranyl ether, a t-butyl ether, anallyl ether, a benzyl ether, a t-butyldimethylsilyl ether, at-butyldiphenylsilyl ether, an acetic acid ester (Ac), ethyl, propyl,butyl, t-butyl or pivalic acid ester and a benzoic acid ester, providedthat at least one of PD₁-PD₄ is not H.

According to some embodiments, each of PD₁-PD₄ is an acetic acid ester(Ac).

According to some embodiments, each of PD₁ and PD₂ is an acetic acidester (Ac).

According to some embodiments, each of PD₁-PD₃ is an acetic acid ester(Ac).

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIGS. 1A, 1B, 1C and 1D present comparative plots of IOP as a functionof time, showing the dose response of α2-inhibitor compounds, accordingto some embodiments of the present invention, in lowering IOP in liverabbits, wherein FIG. 1A shows the results obtained for DiB, FIG. 1Bshows the duration of the effect of DiB while 4AP is added every 2 hoursso as to maintain the raised IOP, FIG. 1C shows the results obtained forDMcP, and FIG. 1D shows the results obtained for DcB;

FIGS. 2A, 2B, 2C and 2D present comparative plots of IOP as a functionof time, demonstrating the capacity of the α2-inhibitor compounds,according to some embodiments of the present invention, to lower IOPbelow basal levels compared to a buffer control when administeredtopically to one eye of a rabbit, while the other eye received PBS as acontrol, wherein FIG. 2A shows the lack of effect of digoxin, FIG. 2Bshows the lack of effect of DiB (a non-cyclic moiety inhibitor), FIG. 2Cshows the notable effect of DMcP, and FIG. 2D shows the notable effectof DcB;

FIGS. 3A, 3B and 3C present comparative plots of IOP as a function oftime, demonstrating the effect of α2-inhibitor compounds, according tosome embodiments of the present invention, to potentiate the drugLatanoprost in lowering IOP below basal levels, wherein FIG. 3A showsthe effect of DcB alone, FIG. 3B shows the effect of Latanoprost alone,and FIG. 3C shows the effect of co-administering DcB with Latanoprost;and

FIG. 4 presents comparative plots of IOP as a function of time,demonstrating the capacity of the trisAcDcB prodrug of the α2-inhibitorcompound DcB, according to some embodiments of the present invention, tolower IOP below basal levels compared to a buffer control whenadministered topically to one eye of a rabbit, while the other eyereceived PBS as a control.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to apharmaceutical agents and, more particularly, but not exclusively, todigoxin and digitoxin derivatives exhibiting selective inhibition ofα2-containing isoforms of Na,K-ATPase, and uses thereof to reduceintraocular pressure (IOP), and/or as cardiotonic agents in a subject inneed thereof.

The principles and operation of the present invention may be betterunderstood with reference to the figures and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

As discussed hereinabove, Na,K-ATPase inhibitors that exhibitselectivity towards tissue-specific isoforms of the protein, presentpharmaceutical advantages such as broader therapeutic window and widerscope of modes of administration. For example, Na,K-ATPase inhibitorsexhibiting selectivity towards protein isoforms containing the α2subunit offer this advantages over unselective inhibitors in treatingmedical conditions wherein lowering the intraocular pressure is calledfor.

While searching for α2-selective inhibitors, the present inventors havesurprisingly found that certain derivatives of digoxin and digitoxin,wherein the perhydro-1-4-oxazepine moiety thereof is N-substituted witha cyclic moiety, exhibit a notable selectivity towards α2-containingisoform of Na,K-ATPase.

Compounds:

According to an aspect of some embodiments of the present inventionthere is provided a compound represented by general Formula I:

wherein:

X is H or OH, whereas derivatives having ×=H are referred to asdigitoxin derivatives, and derivatives having X=—OH are referred to asdigoxin derivatives;

R is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety;

or

R is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is selected from the group consisting of an alkylsulfonyl, anarylsulfonyl and a sulfonamide;

or

R is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkylprovided that at least one of R₁ and R₂ is a C₁-C₄ alkyl, namely B is asecondary amine or tertiary amine.

As used herein, the term “cyclic moiety” refers to a group of atoms thatare covalently attached to one another so as to form at least one ringof atoms. Non-limiting examples of cyclic moieties include unsubstitutedalicyclic moieties, substituted alicyclic moieties, unsubstitutedheterocyclic moieties, substituted heterocyclic moieties, unsubstitutedaryl moieties, substituted aryl moieties, unsubstituted heteroarylmoiety and substituted heteroaryl moieties.

A substituted cyclic moiety has one or more chemical group or atomattached to one of the atoms in the ring of atoms. Examples of suchchemical groups or atoms include, without limitation, C₁-C₆ alkyl,hydroxyl, amine, halo, alkoxy, carboxyl, amide and the like, or a secondcyclic moiety attached by a covalent bond(s) to one or two of the ringatoms of the cyclic moiety.

The terms “hydroxyl” or “hydroxy”, as used herein, refer to an —OHgroup.

As used herein, the term “amine” describes a —NR¹R² group where each ofR¹ and R² is independently hydrogen, alkyl, cycloalkyl, heteroalicyclic,aryl or heteroaryl, as these terms are defined herein.

As used herein, the term “alkyl” describes an aliphatic hydrocarbonincluding straight chain and branched chain groups. The alkyl may have 1to 20 carbon atoms, or 1-10 carbon atoms, and may be branched orunbranched. According to some embodiments of the present invention, thealkyl is a low (or lower) alkyl, having 1-4 carbon atoms (namely,methyl, ethyl, propyl and butyl).

Whenever a numerical range; e.g., “1-10”, is stated herein, it impliesthat the group, in this case the alkyl group, may contain 1 carbon atom,2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbonatoms. In some embodiments, the alkyl is a lower alkyl, including 1-6 or1-4 carbon atoms.

A C₁-C₆ alkyl group refers to any one of the moieties methyl, ethyl,n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, n-pentyl,t-pentyl, neopentyl, i-pentyl, s-pentyl, 3-pentyl, n-hexyl,2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl and 2,3-dimethylbutyl.

The term “halide”, as used herein, refers to the anion of a halo atom,i.e. F⁻, Cl⁻, Br⁻ and I⁻.

The term “halo” refers to F, Cl, Br and I atoms as substituents.

The term “alkoxy” refers to an —OR¹ group, wherein R¹ is as definedherein, but other than hydrogen.

The term “amide” as used herein encompasses C-amide and N-amide.

The term “C-amide” describes a —C(═O)—NR¹R² group, where R¹ and R² areas defined herein.

The term “N-amide” describes a R¹C(═O)—NR²— group, where R¹ and R² areas defined herein.

The term alkylsulfonyl and arylsulfonyl refers to an R¹—S(═O)₂— group,wherein R¹ is as defined herein, but other than hydrogen. Examples ofarylsulfonyl groups include p-toluenesulfonyl (tosyl; Ts),p-bromobenzenesulfonyl (brosyl; Bs), 2- or 4-nitrobenzenesulfonyl(nosyl; Ns), methanesulfonyl (mesyl; Ms), trifluoromethanesulfonyl(triflyl; Tf), and 5-(dimethylamino)naphthalene-1-sulfonyl (Dansyl; Ds).

The term solnfonamide refers to an R³—S(═O)₂— group, wherein R³ is amineas defined herein.

The terms “alicyclic” and “cycloalkyl”, refer to an all-carbonmonocyclic or fused ring (i.e., rings which share an adjacent pair ofcarbon atoms), branched or unbranched group containing 3 or more carbonatoms where one or more of the rings does not have a completelyconjugated pi-electron system, and may further be substituted orunsubstituted. The cycloalkyl can be substituted or unsubstituted.

Examples of alicyclic moieties include, without limitation, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclododecyl.

Examples of substituted alicyclic is selected from the group consistingof 2,3-dimethylcyclopropane-1-yl, 3,3-dimethylcyclobutane-1-yl,3,4-dimethylcyclopentane-1-yl and 3,5-dimethylcyclohexane-1-yl.

The terms “heterocyclic” or “heteroalicyclic”, as used herein, describea monocyclic or fused ring group having in the ring(s) one or more atomssuch as nitrogen, oxygen and sulfur. The rings may also have one or moredouble bonds. However, the rings do not have a completely conjugatedpi-electron system. The heteroalicyclic may be substituted orunsubstituted. Representative examples are morpholine, piperidine,piperazine, tetrahydrofurane, tetrahydropyrane and the like.

In some embodiments, the heterocyclic moiety is selected from the groupconsisting of oxiranyl, aziridinyl, oxetanyl, azetidinyl,tetrahydrofuranyl, pyrrolidinyl, tetrahydropyranyl and piperidinyl.

The term “aryl” describes an all-carbon monocyclic or fused-ringpolycyclic (i.e., rings which share adjacent pairs of carbon atoms)groups having a completely conjugated pi-electron system, as in theexample of phenyl. The aryl group may be unsubstituted or substituted byone or more substituents. Examples of aryls include cyclopentadienyl,phenyl and naphthyl.

The term “heteroaryl” describes a monocyclic or fused ring (i.e., ringswhich share an adjacent pair of atoms) group having in the ring(s) oneor more atoms, such as, for example, nitrogen, oxygen and sulfur and, inaddition, having a completely conjugated pi-electron system. Examples,without limitation, of heteroaryl moieties include pyrrole, furane,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrimidine,quinoline, isoquinoline, purine, thiadiazole, indole and the like. Theheteroaryl group may be unsubstituted or substituted by one or moresubstituents.

According to some embodiments, the spacer moiety A can be a covalentbond, an unsubstituted C₁-C₆ alkyl, a substituted C₁-C₆ alkyl, anunsubstituted C₁-C₆ alkyl interrupted by one or more heteroatom (e.g.,O, N or S) and a substituted C₁-C₆ alkyl interrupted by one or moreheteroatom. A spacer moiety may be substituted with one or more C₁-C₆alkyl, hydroxyl, amine, halo, alkoxy, carboxyl, amide and the like.

In some embodiments, A is a covalent bond and B an alicyclic moiety suchas, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andthe corresponding R in Formula I is:

In some embodiments, A is a covalent bond and B a heteroalicyclic moietysuch as, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and the corresponding R in Formula I is:

In some embodiments, A is —CH₂— and B a cyclic moiety such as, forexample, cyclopropyl, 3,3-dimethylcyclobutane-1-yl and phenyl, and thecorresponding R in Formula I is:

In some embodiments, A is —(CH₂)₂— and B a cyclic moiety such as, forexample, cyclopropyl, and the corresponding R in Formula I is:

In some embodiments, A is —(CH₂)₂— and B a heteroaryl cyclic moiety suchas, for example, imidazolyl, and the corresponding R in Formula I is:

In some embodiment, X is H, A is a covalent bond and B is cyclobutyl.

In some embodiment, X is H, A is —CH₂— and B is cyclopropyl.

In some embodiment, X is OH, A is a covalent bond and B is cyclopropyl,cyclobutyl, cyclopentyl or cyclohexyl.

In some embodiment, X is OH, A is —CH₂— and B is cyclopropyl,3,3-dimethylcyclobutane-1-yl and phenyl.

In some embodiment, X is OH, A is —(CH₂)₂— and B is cyclopropyl.

In some embodiments, R of general Formula I is cyclopropyl,methylcyclopropane, ethylcyclopropane, propylcyclopropane, cyclobutyl,methylcyclobutane, methyl-3,3-dimethylcyclobutane, ethylcyclobutane,propylcyclobutane, cyclopentyl, methylcyclopentane, ethylcyclopentane,propylcyclopentane, cyclohexyl and benzyl. Alternatively, R iscyclopropyl, methylcyclopropane and cyclobutyl.

In some embodiments, A is —CH₂— and B an alkylsulfonyl such as, forexample, methylsulfonyl, and the corresponding R in Formula I is:

In some embodiments, A is —(CH₂)₂— and B an alkylsulfonyl such as, forexample, methylsulfonyl, or a solnfonamide, and the corresponding R inFormula I is:

In some embodiments, A is —(CH₂)₂— and B a tertiary amine such as, forexample, N,N-dimethylamine, and the corresponding R in Formula I is:

In some embodiments, the compound is any one of the structures presentedbelow:

DcP or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclopropyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DMcP or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-(cyclopropylmethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DEcP or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-(2-cyclopropylethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DcB or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclobutyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DcPe or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclopentyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DcHe or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclohexyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DBz or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-benzyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DMDMcB or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-((3,3-dimethylcyclobutyl)methyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DtxMcP or4-((3S,5R,8R,9S,10S,13R,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-(cyclopropylmethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-14-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DtxcB or4-((3S,5R,8R,9S,10S,13R,14S,17R)-3-(((2S,4S,5R,6R)-5-(((2S,4S,5S,6R)-5-((4-cyclobutyl-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-14-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DAz or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-(azetidin-3-yl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DOx or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-3-(((2R,4S,5S,6R)-4-hydroxy-5-(((2S,4S,5S,6R)-4-hydroxy-6-methyl-5-((2-methyl-4-(oxetan-3-yl)-1,4-oxazepan-7-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DTh or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-3-(((2R,4S,5S,6R)-hydroxy-5-(((2S,4S,5S,6R)-4-hydroxy-6-methyl-5-((2-methyl-4-(thietan-3-yl)-1,4-oxazepan-7-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one;and

DHis or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-(2-(1H-imidazol-5-yl)ethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

In some embodiments, the compound is any one of the structures presentedbelow:

DMSM or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-3-(((2S,4S,5R,6R)-4-hydroxy-5-(((2S,4S,5S,6R)-4-hydroxy-6-methyl-5-((2-methyl-4-((methylsulfonyl)methyl)-1,4-oxazepan-7-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

DESM or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-3-(((2S,4S,5R,6R)-4-hydroxy-5-(((2S,4S,5S,6R)-4-hydroxy-6-methyl-5-((2-methyl-4-(2-(methylsulfonyl)ethyl)-1,4-oxazepan-7-yl)oxy)tetrahydro-2H-pyran-2-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one;and

DESA or2-(7-(((2R,3S,4S,6S)-6-(((2R,3R,4S,6S)-6-(((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl)oxy)-4-hydroxy-2-methyltetrahydro-2H-pyran-3-yl)oxy)-2-methyl-1,4-oxazepan-4-yl)ethanesulfonamide

In some embodiments, the compound is any one of the structures presentedbelow:

DEDA or4-((3S,5R,8R,9S,10S,12R,13S,14S,17R)-3-(((2R,4S,5S,6R)-5-(((2S,4S,5S,6R)-5-((4-(2-(dimethylamino)ethyl)-2-methyl-1,4-oxazepan-7-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-12,14-dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)furan-2(5H)-one

The present embodiments further encompass any enantiomers,diastereomers, optical isomers, prodrugs, solvates, hydrates,polymorphs, geometrical isomers and/or pharmaceutically acceptable saltsof the compounds described herein.

Any one or more of the compounds presented herein may be present as asalt. The term “salt” encompasses both basic and acid addition salts,and include salts formed with organic and inorganic anions and cations.The term “organic or inorganic cation” refers to counter-ions for anacid. The counter-ions can be chosen from the alkali and alkaline earthmetals, (such as lithium, sodium, potassium, barium, aluminum andcalcium), ammonium and the like. Furthermore, the term includes saltsthat form by standard acid-base reactions of basic groups and organic orinorganic acids. Such acids include hydrochloric, hydrofluoric,hydrobromic, trifluoroacetic, sulfuric, phosphoric, acetic, succinic,citric, lactic, maleic, fumaric, cholic, pamoic, mucic, D-camphoric,phthalic, tartaric, salicylic, methanesulfonic, benzenesulfonic,p-toluenesulfonic, sorbic, picric, benzoic, cinnamic, and like acids.

As used herein, the phrase “pharmaceutically acceptable salt” refers toa charged species of the parent compound and its counter-ion, which istypically used to modify the solubility characteristics of the parentcompound and/or to reduce any significant irritation to an organism bythe parent compound, while not abrogating the biological activity andproperties of the administered compound. A pharmaceutically acceptablesalt of a compound as described herein can alternatively be formedduring the synthesis of the compound, e.g., in the course of isolatingthe compound from a reaction mixture or re-crystallizing the compound.

In the context of some of the present embodiments, a pharmaceuticallyacceptable salt of the compounds described herein may optionally be anacid addition salt comprising at least one basic (e.g., amine and/orguanidine) group of the compound which is in a positively charged form(e.g., wherein the basic group is protonated), in combination with atleast one counter-ion, derived from the selected base, that forms apharmaceutically acceptable salt.

The acid addition salts of the compounds described herein may thereforebe complexes formed between one or more basic groups of the compound andone or more equivalents of an acid.

Depending on the stoichiometric proportions between the charged group(s)in the compound and the counter-ion in the salt, the acid additionssalts can be either mono-addition salts or poly-addition salts.

The phrase “mono-addition salt”, as used herein, refers to a salt inwhich the stoichiometric ratio between the counter-ion and charged formof the compound is 1:1, such that the addition salt includes one molarequivalent of the counter-ion per one molar equivalent of the compound.

The phrase “poly-addition salt”, as used herein, refers to a salt inwhich the stoichiometric ratio between the counter-ion and the chargedform of the compound is greater than 1:1 and is, for example, 2:1, 3:1,4:1 and so on, such that the addition salt includes two or more molarequivalents of the counter-ion per one molar equivalent of the compound.

An example, without limitation, of a pharmaceutically acceptable saltwould be an ammonium cation or guanidinium cation and an acid additionsalt thereof.

The acid addition salts may include a variety of organic and inorganicacids, such as, but not limited to, hydrochloric acid which affords ahydrochloric acid addition salt, hydrobromic acid which affords ahydrobromic acid addition salt, acetic acid which affords an acetic acidaddition salt, ascorbic acid which affords an ascorbic acid additionsalt, benzenesulfonic acid which affords a besylate addition salt,camphorsulfonic acid which affords a camphorsulfonic acid addition salt,citric acid which affords a citric acid addition salt, maleic acid whichaffords a maleic acid addition salt, malic acid which affords a malicacid addition salt, methanesulfonic acid which affords a methanesulfonicacid (mesylate) addition salt, naphthalenesulfonic acid which affords anaphthalenesulfonic acid addition salt, oxalic acid which affords anoxalic acid addition salt, phosphoric acid which affords a phosphoricacid addition salt, toluenesulfonic acid which affords ap-toluenesulfonic acid addition salt, succinic acid which affords asuccinic acid addition salt, sulfuric acid which affords a sulfuric acidaddition salt, tartaric acid which affords a tartaric acid addition saltand trifluoroacetic acid which affords a trifluoroacetic acid additionsalt. Each of these acid addition salts can be either a mono-additionsalt or a poly-addition salt, as these terms are defined herein.

As used herein, the term “enantiomer” refers to a stereoisomer of acompound that is superposable with respect to its counterpart only by acomplete inversion/reflection (mirror image) of each other. Enantiomersare said to have “handedness” since they refer to each other like theright and left hand. Enantiomers have identical chemical and physicalproperties except when present in an environment which by itself hashandedness, such as all living systems. In the context of the presentembodiments, a compound may exhibit one or more chiral centers, each ofwhich exhibiting an R- or an S-configuration and any combination, andcompounds according to some embodiments of the present invention, canhave any their chiral centers exhibit an R- or an S-configuration.

The term “diastereomers”, as used herein, refers to stereoisomers thatare not enantiomers to one another. Diastereomerism occurs when two ormore stereoisomers of a compound have different configurations at one ormore, but not all of the equivalent (related) stereocenters and are notmirror images of each other. When two diastereoisomers differ from eachother at only one stereocenter they are epimers. Each stereo-center(chiral center) gives rise to two different configurations and thus totwo different stereoisomers. In the context of the present invention,embodiments of the present invention encompass compounds with multiplechiral centers that occur in any combination of stereo-configuration,namely any diastereomer.

All stereoisomers, optical and geometrical isomers of the compounds ofthe present invention are contemplated, either in admixture or in pureor substantially pure form. The compounds of the present invention canhave asymmetric centers at one or more of the atoms. Consequently, thecompounds can exist in enantiomeric or diastereomeric forms or inmixtures thereof. The present invention contemplates the use of anyracemates (i.e. mixtures containing equal amounts of each enantiomers),enantiomerically enriched mixtures (i.e., mixtures enriched for oneenantiomer), pure enantiomers or diastereomers, or any mixtures thereof.The chiral centers can be designated as R or S or R,S or d,D, l,L ord,l, D,L.

The term “prodrug” refers to an agent, which is converted into theactive compound (the active parent drug) in vivo. Prodrugs are typicallyuseful for facilitating the administration of the parent drug. They may,for instance, be bioavailable by oral administration whereas the parentdrug is not. A prodrug may also have improved solubility as comparedwith the parent drug in pharmaceutical compositions. Prodrugs are alsooften used to achieve a sustained release of the active compound invivo. An example, without limitation, of a prodrug would be a compoundof the present invention, having one or more carboxylic acid moieties,which is administered as an ester (the “prodrug”). Such a prodrug ishydrolyzed in vivo, to thereby provide the free compound (the parentdrug). The selected ester may affect both the solubility characteristicsand the hydrolysis rate of the prodrug.

The term “solvate” refers to a complex of variable stoichiometry (e.g.,di-, tri-, tetra-, penta-, hexa-, and so on), which is formed by asolute (the compound of the present invention) and a solvent, wherebythe solvent does not interfere with the biological activity of thesolute. Suitable solvents include, for example, ethanol, acetic acid andthe like.

The term “hydrate” refers to a solvate, as defined hereinabove, wherethe solvent is water.

The present invention also includes solvates of the compounds of thepresent invention and salts thereof. “Solvate” means a physicalassociation of a compound of the invention with one or more solventmolecules. This physical association involves varying degrees of ionicand covalent bonding, including hydrogen bonding. In certain instancesthe solvate will be capable of isolation. “Solvate” encompasses bothsolution-phase and isolatable solvates. Non-limiting examples ofsuitable solvates include ethanolates, methanolates and the like.“Hydrate” is a solvate wherein the solvent molecule is water.

The present invention also includes polymorphs of the compounds of thepresent invention and salts thereof. The term “polymorph” refers to aparticular crystalline state of a substance, which can be characterizedby particular physical properties such as X-ray diffraction, IR spectra,melting point, and the like.

According to some aspects of some embodiments of the present invention,the compound is represented by general Formula III:

wherein:

X is H or OH;

R′ is selected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆haloalkyl (C₁-C₆ alkyl substituted with at least one halo),—(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂,—(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂and —(CR^(b)R^(c))n-NH₂;

Y is O or S;

R₁, R₂ and R₃ are each independently H or a C₁-C₄ alkyl;

Ra is a C₁-C₄ alkyl;

R^(b), R^(c), R^(d) and R^(e) are each independently selected from H, aC₁-C₄ alkyl and a C₁-C₄ hydroxyalkyl; and

n is 0, 1 or 2;

or

R′ is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is a cyclic moiety;

or

R′ is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is selected from the group consisting of an alkylsulfonyl, anarylsulfonyl and a sulfonamide;

or

R′ is represented by general Formula II:

the wiggled line represents the N-link to the compound;

A is a spacer moiety or a covalent bond; and

B is —NR₁R₂, wherein R₁ and R₂ are each independently H or a C₁-C₄ alkylprovided that at least one of R₁ and R₂ is a C₁-C₄ alkyl, including anypharmaceutically acceptable salt, prodrug, hydrate, solvate, enantiomerand diastereomer thereof, and any mixtures thereof, and apharmaceutically acceptable carrier.

Additional alternative structures of R′ are described in WO 2015/029035,which is hereby specifically incorporated by reference as if fully setforth herein, thereby describing each and all such additionalalternative structures.

Process of Preparing the Compounds:

According to an aspect of some embodiments of the present invention,there is provided a process for preparing the compounds represented bygeneral Formula I, the process includes:

converting the third digitoxose moiety of digoxin or digitoxin into adialdehyde; and

reacting said dialdehyde with a reagent represented by general formulaIV:B-A-NH₂   Formula IV

A is a spacer moiety or a covalent bond as described hereinabove; and

B is selected form the group consisting of a cyclic moiety, analkylsulfonyl, arylsulfonyl, a sulfonamide, a secondary amine and atertiary amine, as described hereinabove.

As can be seen in Scheme 1, the conversion of the third digitoxosemoiety of digoxin or digitoxin is typically effected by selectiveoxidation thereof into a dialdehyde by reacting digoxin or digitoxinwith a reagent that breaks apart 1,2-diols (vicinal diols) to affordaldehydes and/or ketones, such as sodium periodate (NaIO₄); The reactionof the dialdehyde is effected by reductive amination of the dialdehydeusing a free amine of R (R—NH₂) in the presence of, e.g., NaCNBH₃.

Selective Affinity and Inhibition:

The results presents in the Examples section below (see, e.g., Table 2)show clearly that compounds encompassed by general Formula I, accordingto embodiments of the present invention, exhibit high selectivity forα2β3 over α1β1 compared to digoxin and digitoxin. The results obtainedfor cyclic R-substituents (R comprising a cyclic moiety), some with 4carbon atoms, provide a clear indication for selective structuralinteractions of the modified sugar of digoxin with the 33 subunit. Thiseffect may also be combined with an enhanced interaction with the α2subunit. The detailed enzymological study, presented hereinbelow, hasbeen obtained with the purified detergent-soluble human Na,K-ATPaseisolated isoform complexes, as well as with intact bovine ciliary NPEcells, i.e., selective inhibition of human Na,KATPase isolated isoformcomplexes provided corroborating indication of inhibition by thecompounds provided herein also of isoform mixture (α1β1 plus α2β3) asthese are expressed in native NPE cell membranes.

According to some embodiments, the compounds presented herein, such as acompound represented by general Formula I or by general Formula III,exhibit an affinity to at least one isoform of Na,K-ATPase.

In the context of embodiments of the present invention, isoforms ofNa,K-ATPase include any combination of α1, α2, α3 and α4 complexed withβ1, β2 and β3; hence, isoforms of Na,K-ATPase include α1β1, α1β2, α1β3,α2β1, α2β2, α2β3, α3β1, α3β2, α3β3, α4β1, α4β2 and α4β3.

According to some embodiments of the present invention, a compoundrepresented by general Formula I or by general Formula III, has a higheraffinity to isoforms containing an α2 subunit, compared to its affinityto isoforms containing of the a subunits, such as α1, as demonstrated inthe Examples section that follows below.

The phrase “higher affinity to”, as used herein, is a relative term thatmeans that a given ligand molecule (compound, inhibitor, drug, etc.) isattracted and can bind to and (form a) complex with a target entity(protein, enzyme, drug-target) more strongly compared to its binding toanother target entity. Affinity can be measured directly and indirectlyby a number of methodologies. In some embodiments of the presentinvention, the affinity is referred to in terms of Ki (inhibitionconstant) or K_(D) (dissociation constant), as these terms are known inthe art. For example, a small value for Ki means that an inhibitor has ahigher effective affinity to the enzyme relative to an affinity of thesame inhibitor to another enzyme or another inhibitor to the sameenzyme.

According to some embodiments of the present invention, a compoundrepresented by general Formula I or by general Formula III, is aselective inhibitor of α2-containing isoforms of Na,K-ATPase, comparedto α1-containing isoforms of Na,K-ATPase, as demonstrated in theExamples section that follows below.

The terms “selective inhibitor of α2-containing isoforms of Na,K-ATPase”or “selective inhibitor of the α2 isoform of Na,K-ATPase” or“α2-selective inhibitor”, as used herein interchangeably, refer to acompound that inhibits α2-containing isoforms of Na,K-ATPase to agreater degree than the compound inhibits other isoforms of Na,K-ATPase,such as those containing α1. In some embodiments, the compoundsdescribed herein are selective for the α2β1, α2β2 and/or α2β3 isoformsof Na,K-ATPase over the α1β3 isoform thereof. In some embodiments, theselectivity of the compound for the α2-containing isoform of Na,K-ATPase(e.g., α2β1, α2β2 and/or α2β3 isoform) is at least about 4-fold (300%more selective) over other isoforms, or at least 5-fold (400%), at least6-fold (500%), at least 8-fold (700%), at least 10-fold, at least16-fold, at least 20-fold, at least 30-fold, or at least 50-fold greaterinhibition of the α2-containing isoform of Na,K-ATPase over otherisoforms of Na,K-ATPase.

Uses of α2-Selective Inhibitors:

The experimental work presented in the Examples section belowdemonstrates a clear correlation between increased α2β3-selectivity ofthe compounds presented herein and potency and duration in reducing IOPof either pharmacologically raised or basal (normal) IOP. Thus theresults support a central role of α2β3 in production of aqueous humour.The mechanism of the IOP reduction has been shown to involve inhibitionof active Na and K fluxes via NPE cells and reduction of inflow ofaqueous humour after topical administration of compound(s) andpermeation thereof via the cornea.

Without being bound by any particular theory, it is assumed that the lowKi values for inhibition of α2β3 (Ki of about 4 nM) and hydrophobicproperties of the compounds according to some embodiments of the presentinvention, suggest that both traits contribute to the potency and longduration of action exhibited by the compounds provided herein. Thefinding that the compounds provided herein are effective in reducingbasal (normal) IOP as low as 25-30% (see, e.g., FIGS. 2A-D and FIGS.3A-3C), as well as the reduction of acute 4AP-induced raised IOP (see,e.g., FIGS. 1A-1D), provides an insight into the mechanism of action ofthe compounds.

The compounds presented herein are useful as topical opthalmologicalagents for treatment of glaucoma, since they exhibit at least one ofefficacy and low levels of side-effects. In relations to alternativedrugs, the compounds presented herein are useful as topicalopthalmological agents for treatment of glaucoma, since they have beenshown to exhibit at least one of improved efficacy, extended duration ofdesired effects and reduced side-effects, compared to currentlyavailable drugs, exemplified in the Examples section that follows belowby the first-line drug latanoprost, and iopidine, used in short-termadjunctive therapy of chronic glaucoma. In general, currently availabledrugs include β-adrenergic antagonists and carbonic anhydraseinhibitors, that reduce the rate of aqueous humor production, orprostaglandin analogs, cholinergic agonists and sympathomimetics, thatincrease the rate of outflow through the trabecular meshwork anduveoscleral pathway. In this respect, the experimental results presentedhereinbelow, show 25-30% higher reduction in basal IOP effected by thecompounds presented herein in the rabbits, compared to Latanoprost, thecurrent first-line drug for treatment of glaucoma.

In principle, the ability of the compounds presented herein, accordingto some embodiments, to reduce the basal IOP, could be relevant not onlyto optical hypertension and primary open angle glaucoma but also tonormotensive glaucoma for which reduction of IOP below the basal levelis required.

Regarding local toxicity, the rationale for making α2β3-selectiveinhibitors included not only the expectation of high potency but also ofminimal local side-effects. In the Examples section below it is shownthat corneal swelling, used to assess such adverse effect, is notobserved at least over several days of topical administration of thecompounds presented herein (see, e.g., Table 4). Similar evidence forsafety was observed by inspection of local redness and irritation.

Concerning possible systemic toxicity of the α2β3-selective compoundspresented herein, it is expected that cardiotoxicity, such as isassociated with clinical use of digoxin, would be minimal, because theα2-selective compounds are likely to be intrinsically non-cardiotoxic,compared to a non-selective cardiac glycoside, and also because thepharmacokinetic concentration is likely to be low (see, e.g., inhibitionresults in Table 2).

The results presented hereinbelow indicate that after repeatedadministration over several weeks of the compounds according toembodiments of the present invention, no signed of adverse effects havebeen observed, indicating that compounds represented by general FormulaI or III are not toxic and exhibit essentially no adverse effects.

Thus, the α2-selective inhibitors presented herein (compoundsrepresented by general Formula I or by general Formula III) can be usedto reduce intraocular pressure in a subject in need thereof, whiletaking advantage of the selective affinity these inhibitors exhibittowards α2-containing isoforms of Na,K-ATPase, which minimize adverseeffects associated with unselective inhibition of the other isoforms.

According to an aspect of some embodiments of the present invention,there is provided a pharmaceutical composition which includes as anactive ingredient any one of the compounds represented by generalFormula I and a pharmaceutically acceptable carrier.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare: propylene glycol, saline, emulsions and mixtures of organicsolvents with water, as well as solid (e.g., powdered) and gaseouscarriers.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the compounds presentedherein into preparations which, can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

According to some embodiments, the administration is effected topically,extraocularly, intraocularly and/or intravitreally. In some embodiments,the pharmaceutical composition is formulated as an ophthalmiccomposition suitable for topical, extraocular, intraocular and/orintravitreal administration to the eye of the subject. According to someembodiments, the pharmaceutical composition of the invention is anophthalmic composition which is administered topically onto the eye of asubject for facilitating effective intraocular levels of the compoundand for preventing unnecessary and unintentional levels of the compoundin other tissues and/or organs. Such a non-systemic, site-specificadministration reduces the side effects associated with the compounds.

In the context of some embodiments of the present invention, topicaland/or extraocular administration is effected by applying thecompound(s), or compositions and medicaments comprising the compound(s)to the eye or a bodily surface near the eye. According to someembodiments, the composition may take the form of an eye-drop solution,a spray, an eye wash solution, an ointment, a lotion, a suspension, agel or a cream. The topical pharmaceutical compositions may be in theform of eye-drops to be applied by instillation into the eye or may bein the form of a viscous ointment, gel or cream to be applied by anointment onto the ocular surface and may contain control release meansfor facilitating sustained release over a prolonged period of time.

In the context of some embodiments of the present invention, intraocularand/or intravitreal administration is effected by injecting thecompound(s), or compositions and medicaments comprising the compound(s)into the eye or into a bodily tissue near the eye. According to someembodiments, the composition may take the form of an injectablesolution.

According to some embodiments, oral or otherwise systemic administrationin a dosage effective for reducing the intraocular pressure is alsopossible. For example, the composition may be administered by a dermalpatch for extended release.

According to some embodiments, the administration is effected orally.For oral administration, the compounds presented herein can beformulated readily by combining the compounds with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompounds presented herein to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for oral ingestion by a patient. Pharmacological preparations for oraluse can be made using a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/orphysiologically acceptable polymers such as polyvinylpyrrolidone (PVP).If desired, disintegrating agents may be added, such as cross-linkedpolyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such assodium alginate.

Pharmaceutical compositions, which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, thecompounds presented herein may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for the chosen routeof administration.

For injection, the compounds presented herein may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline bufferwith or without organic solvents such as propylene glycol, polyethyleneglycol.

For transmucosal administration, penetrants are used in the formulation.Such penetrants are generally known in the art.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active aminoglicoside compounds doses.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds presented herein areconveniently delivered in the form of an aerosol spray presentation(which typically includes powdered, liquefied and/or gaseous carriers)from a pressurized pack or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compounds presented herein and a suitablepowder base such as, but not limited to, lactose or starch.

The compounds presented herein may be formulated for parenteraladministration, e.g., by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampoules or in multidose containers with optionally, an addedpreservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the compounds preparation in water-soluble form.Additionally, suspensions of the compounds presented herein may beprepared as appropriate oily injection suspensions and emulsions (e.g.,water-in-oil, oil-in-water or water-in-oil in oil emulsions). Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran.

Optionally, the suspension may also contain suitable stabilizers oragents, which increase the solubility of the compounds presented hereinto allow for the preparation of highly concentrated solutions.

Alternatively, the compounds presented herein may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free water,before use.

The compounds presented herein may also be formulated in rectalcompositions such as suppositories or retention enemas, using, e.g.,conventional suppository bases such as cocoa butter or other glycerides.

The pharmaceutical compositions herein described may also comprisesuitable solid of gel phase carriers or excipients. Examples of suchcarriers or excipients include, but are not limited to, calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin and polymers such as polyethylene glycols.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofcompounds presented herein effective to prevent, alleviate or amelioratesymptoms of the disorder, or prolong the survival of the subject beingtreated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any compounds presented herein used in the methods of the presentembodiments, the therapeutically effective amount or dose can beestimated initially from activity assays in animals. For example, a dosecan be formulated in animal models to achieve a circulatingconcentration range that includes the mutation suppression levels asdetermined by activity assays (e.g., the concentration of the testcompounds which achieves a substantial read-through of the truncationmutation). Such information can be used to more accurately determineuseful doses in humans.

Toxicity and therapeutic efficacy of the compounds presented herein canbe determined by standard pharmaceutical procedures in experimentalanimals, e.g., by determining the EC₅₀ (the concentration of a compoundwhere 50% of its maximal effect is observed) and the LD₅₀ (lethal dosecausing death in 50% of the tested animals) for a subject compound. Thedata obtained from these activity assays and animal studies can be usedin formulating a range of dosage for use in human.

The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition. (See e.g., Fingl et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the compounds presented herein which are sufficient tomaintain the desired effects, termed the minimal effective concentration(MEC). The MEC will vary for each preparation, but can be estimated fromin vitro data; e.g., the concentration of the compounds necessary toachieve 50-90% expression of the whole gene having a truncationmutation, i.e. read-through of the mutation codon. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. HPLC assays or bioassays can be used to determine plasmaconcentrations.

Dosage intervals can also be determined using the MEC value.Preparations should be administered using a regimen, which maintainsplasma levels above the MEC for 10-90% of the time, preferable between30-90% and most preferably 50-90%.

Depending on the severity and responsiveness of the chronic condition tobe treated, dosing can also be a single periodic administration of aslow release composition described hereinabove, with course of periodictreatment lasting from several days to several weeks or until sufficientamelioration is effected during the periodic treatment or substantialdiminution of the disorder state is achieved for the periodic treatment.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc. Compositions of the present invention may, if desired, be presentedin a pack or dispenser device, such as an FDA (the U.S. Food and DrugAdministration) approved kit, which may contain one or more unit dosageforms containing the active ingredient. The pack may, for example,comprise metal or plastic foil, such as, but not limited to a blisterpack or a pressurized container (for inhalation). The pack or dispenserdevice may be accompanied by instructions for administration. The packor dispenser may also be accompanied by a notice associated with thecontainer in a form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals, which notice is reflectiveof approval by the agency of the form of the compositions for human orveterinary administration.

Such notice, for example, may be of labeling approved by the U.S. Foodand Drug Administration for prescription drugs or of an approved productinsert. Compositions comprising a compound according to the presentembodiments, formulated in a compatible pharmaceutical carrier may alsobe prepared, placed in an appropriate container, and labeled fortreatment of an indicated condition or diagnosis, as is detailedhereinabove.

In accordance with other embodiments, the compounds presented herein maybe loaded into a drug-delivery device to be inserted or implanted intothe eye of the subject for allowing releasing of the compound in acontrolled and continuous rate, by dissolving, diffusion or leaching,thus maintaining effective therapeutic concentration over a prolongedperiod of time. The drug-delivery device may be for example abiocompatible thin film loaded with the active agent, inserted forexample beneath the lower eyelid. In some embodiments, the drug-deliverydevice is a contact lens or any other ophthalmic device, as these areknown in the art.

In some embodiments, the pharmaceutical composition is packaged in apackaging material and identified in print, or on the packagingmaterial, for use in reducing intraocular pressure (IOP).

According to an aspect of some embodiments of the present invention,there is provided a use of the compounds represented by general FormulaI, or a pharmaceutical composition comprising the same, for themanufacture of a medicament for reducing intraocular pressure (IOP).

According to an aspect of some embodiments of the present invention,there is provided a method of reducing intraocular pressure (IOP) in asubject, the method includes administering to a subject in need thereofa therapeutically effective amount of a compound represented by generalFormula I, or a pharmaceutical composition comprising the same.

According to some embodiments of the present invention, intraocularpressure (IOP) is associated with glaucoma, low-tension glaucoma andnormal-tension glaucoma. Hence, the compounds, compositions andmedicaments presented herein are useful in treating, without limitation,glaucoma, low-tension glaucoma and normal-tension glaucoma.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

As used herein, the phrase “therapeutically effective amount” describesan amount of the polymer being administered which will relieve to someextent one or more of the symptoms of the condition being treated.

In some embodiments, the concentration of the compounds presented hereinin the pharmaceutical compositions or medicaments presented herein, isin the range of about 1 to about 5,000 μg/ml of composition, preferablyfrom about 80 to about 800 ag/ml and the formulation is preferablyapplied in one to four doses per day wherein each dose contains about 1to 125 μg of the compound, or from about 2 to about 20 μg of thecompound.

Co-Administration with Other IOP Reducing Agents:

In the experimental section presented below, it has been demonstratedthat a combination of a compound according to some embodiments of thepresent invention and Latanoprost resulted in a notable increase in theduration of the effects for the combination compared to the individuallyadministered agents.

In the context of some embodiments of the present invention, an activeagent which is not a compound represented by general Formula I orgeneral Formula III, is referred to herein as “another agent” or “otheragent”.

According to an aspect of some embodiments of the present invention,there is provided a pharmaceutical composition which includes as activeingredients at least one other agent (active ingredient) which is in usein reducing IOP and is not a compound represented by general Formula Ior general Formula III, a compound represented by general Formula I orgeneral Formula III, and a pharmaceutically acceptable carrier.

Non-limiting examples of active ingredients which are in use in reducingIOP and which are referred to herein other agents, include prostaglandinanalogs, β-blockers, adrenergic agents, α2-adrenergic receptor agonists,miotic agents, carbonic anhydrase inhibitors and cholinergic agonists.

Non-limiting examples of prostaglandin analogs include Latanoprost(Xalatan), Bimatoprost (Lumigan) and Travoprost (Travatan).

Non-limiting examples of β-blockers include Timolol (Timoptic) andBetaxolol (Betoptic).

A non-limiting example of adrenergic agents is Brimonidine (Alphagan).

A non-limiting example of miotic agents is Pilocarpine (Isoptocarpine,Pilocar).

Non-limiting examples of carbonic anhydrase inhibitors includeDorzolamide (Trusopt), Brinzolamide (Azopt) and Acetazolamide (Diamox).

Non-limiting examples of cholinergic agonists include carbachol(Miostat), echothiophate (Phospholine) and pilocarpine (Isopto Carpine,Pilopine).

Administration of more than one active agent to a subject is generallyknown in the art as co-administration. The term “co-administration” asused herein, refers to a concomitant administration of more than oneactive agent (active ingredient) to a subject, whereas in the context ofembodiments presented herein, the term “concomitant” means that theco-administered active agents are present in the subject (PK), orotherwise exert an effect (PD), at similar, identical, consecutive orpartially overlapping periods of time.

In the context of co-administration of more than one active agent, theterms “substantially simultaneous” and “rapid succession” correspond tothe term “concomitant” as used herein, namely meaning that the period oftime between a first administration and a second administration of morethan one active agent is sufficiently short to be regarded as a singleadministration event, and/or a number of administrations of differentactive agents takes place within 5-60 minutes or less. Optionally, eachadministration in such “rapid succession” delivers to the user adifferent amount or composition of one or more pharmaceutically activeagents. Alternatively, two or more of the administrations provide thesame composition and amount of the one or more pharmaceutically activeagents. In some embodiments, the later administration of the secondactive agent is performed at such timing that the first active agent ofa previous administration within the same rapid succession does not yethave a significant pharmacodynamic effect or before it can be measured(pharmacokynetically) by, e.g., blood concentration thereof the firstactive agent.

According to some embodiments of the present invention, theco-administration of at least one other agent which is in use inreducing IOP and is not a compound represented by general Formula I orgeneral Formula III, and a compound represented by general Formula I orgeneral Formula III, exhibits a potentiating effect, namely that theeffect of the co-administered active ingredients is greater in at leastone parameter, such as magnitude or duration, compared to the effectsexhibited by each of the active ingredients when administered alone(separately).

Accordingly, there is provided a use of at least one other agent whichis in use in reducing IOP, and a compound represented by general FormulaI or general Formula III, for the manufacture of a medicament forreducing IOP.

According to an aspect of some embodiments of the present invention,there is provided a method of reducing IOP in a subject in need thereof,the method includes co-administering to the subject a therapeuticallyeffective amount of at least one other agent which is in use in reducingIOP, and a therapeutically effective amount of a compound represented bygeneral Formula I or general Formula III.

According to an aspect of some embodiments of the present invention,there is provided a method of reducing IOP in a subject in need thereof,the method includes co-administering to the subject a synergisticallyeffective amount of at least one other agent which is in use in reducingIOP, and a synergistically effective amount of a compound represented bygeneral Formula I or general Formula III. It is noted herein that asynergistically effective amount is also a therapeutically effectiveamount in the sense of providing the desired therapeutic effect, and issmaller than the therapeutically effective amount of asingly-administered active ingredient.

In the context of the herein provided uses and method ofco-administration of other agents and the compounds presented herein,the other agent is a prostaglandin analog.

In the context of the herein provided uses and method ofco-administration of other agents and the compounds presented herein,the other agent is a β-blocker.

In the context of the herein provided uses and method ofco-administration of other agents and the compounds presented herein,the other agent is an adrenergic agent.

In the context of the herein provided uses and method ofco-administration of other agents and the compounds presented herein,the other agent is an α2-adrenergic receptor agonist.

In the context of the herein provided uses and method ofco-administration of other agents and the compounds presented herein,the other agent is a miotic agent.

In the context of the herein provided uses and method ofco-administration of other agents and the compounds presented herein,the other agent is a carbonic anhydrase inhibitor.

In the context of the herein provided uses and method ofco-administration of other agents and the compounds presented herein,the other agent is a cholinergic agonist.

Cardiotonic Agent:

According to some embodiments, the selectivity of the compoundspresented herein can be utilized in the treatment of other medicalconditions. For example, an α2-selective inhibitor, such as thecompounds presented herein and represented by general Formula I, can beused as an effective cardiotonic agent, with reduced cardiotoxicity,compared to known agents such as digoxin.

According to an aspect of embodiments of the present invention, there isprovided a method of treating a heart condition which is carried out byadministering to a subject in need thereof a therapeutically effectiveamount of a compound represented by general Formula I.

According to an aspect of embodiments of the present invention, there isprovided a pharmaceutical composition which includes as an activeingredient a compound represented by general Formula I and apharmaceutically acceptable carrier, identified in print, or on apackaging material, for use in a treatment of a heart condition.

According to an aspect of embodiments of the present invention, there isprovided a uses of a compound represented by general Formula I or apharmaceutical composition comprising the same, for the manufacture of amedicament for treating a heart condition.

Examples of heart conditions which are relevant in the context ofembodiments of the resent invention, include, without limitation, atrialfibrillation, atrial flutter, mitral stenosis, chronic heart failure andcongestive heart failure.

In some embodiments, the present invention provides cardiotoniccompositions comprising a therapeutically effective amount of thecompounds represented by general Formula I, or a pharmaceuticalcomposition comprising the same. In accordance with such embodiments,the compounds may be formulated for oral, buccal, topical, intravenous,parenteral or rectal administration.

A compound represented by general Formula I or a compound represented bygeneral Formula III can be used according to embodiments the presentinvention to treat a heart condition in combination with one or moreother drugs for treating a heart a condition, such as, but not limitedto, selective and nonselective β-blocker agents, anticoagulation agents,angiotensin-converting-enzyme inhibitors (ACE inhibitors) andangiotensin II receptor antagonists.

According to an aspect of embodiments of the present invention, there isprovided a method of treating a heart condition in a subject in needthereof, which includes co-administering to the subject atherapeutically effective amount of an agent selected from the groupconsisting of a β-blocker, an anticoagulation agent, anangiotensin-converting-enzyme inhibitor and an angiotensin II receptorantagonist; and

a compound represented by Formula III, as these are described herein,and a pharmaceutically acceptable carrier.

According to an aspect of embodiments of the present invention, there isprovided a pharmaceutical composition that includes, as activeingredients, at least one ingredient selected from the group consistingof a β-blocker, an anticoagulation agent, anangiotensin-converting-enzyme inhibitor and an angiotensin II receptorantagonist; and a compound represented by Formula III, as these aredescribed herein.

According to an aspect of embodiments of the present invention, there isprovided a use of an agent selected from the group consisting of aβ-blocker, an anticoagulation agent, an angiotensin-converting-enzymeinhibitor and an angiotensin II receptor antagonist, and a compoundrepresented by Formula III, as these are described herein, for themanufacture of a medicament for treating a heart condition.

Nonselective β-blocker agents include propranolol, bucindolol,carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutolol,pindolol, sotalol, timolol, eucommia bark (herb); β1-selective agentsinclude acebutolol, atenolol, betaxolol, bisoprolol, celiprolol,esmolol, metoprolol, nebivolol; β2-selective agents include butaxamineand ICI-118,551; and β3-selective agents include SR 59230A.

Anticoagulation agents include heparin, dicumarol, coumadin (warfarin)and aspirin.

ACE inhibitors include captopril, zofenopril, enalapril (vasotec,renitec), ramipril (altace, prilace, ramace, ramiwin, triatec, tritace),quinapril (accupril), perindopril (coversyl, aceon, perindo), lisinopril(listril, lopril, novatec, prinivil, zestril), benazepril (lotensin),imidapril (tanatril), trandolapril (mavik, odrik, gopten), cilazapril(inhibace) and fosinopril (fositen, monopril).

Angiotensin II receptor antagonists include losartan, EXP 3174,candesartan, valsartan, irbesartan, telmisartan, eprosartan, olmesartan,azilsartan and fimasartan.

According to some embodiments, the compound represented by generalFormula I or the compound represented by general Formula III, accordingto embodiments of the present invention, and any one of the other drugsfor treating a heart a condition can be co-formulated in a singlecomposition, or be formulated into individual compositions.

High-Risk Pharmacokinetics:

When plasma concentrations of active drug depend exclusively on a singlemetabolic pathway, any condition that inhibits that pathway (be itdisease-related, genetic, or due to a drug interaction) can lead todramatic changes in drug concentrations and marked variability in drugaction. This problem of high-risk pharmacokinetics is especiallypronounced in drug elimination that relies on a single pathway. In thiscase, inhibition of the elimination pathway leads to striking elevationof drug concentration. For drugs with a narrow therapeutic window, thisleads to an increased likelihood of dose-related toxicity. An example isdigoxin, whose elimination is dependent on P-glycoprotein; many drugsinhibit P-glycoprotein activity (amiodarone, quinidine, erythromycin,cyclosporine, itraconazole) and coadministration of these with digoxinreduces digoxin clearance, and increases toxicity unless maintenancedoses are lowered. Drugs with a high risk of generating pharmacokineticinteractions with digoxin include antacids and bile acid sequestrants,which can cause reduced absorption; inhibitors of CYPs and ofP-glycoprotein such as amiodarone, quinidine, amiodarone, verapamil,cyclosporine, itraconazole and erythromycin which can cause decreasedclearance.

Since compounds encompassed under Formula III are in essence digoxinderivatives that exhibit lower digoxin toxicity, these compounds can beused with any of the drugs described above which exhibit adversedrug-drug interaction with digoxin.

According to an aspect of embodiments of the present invention, there isprovided use of the compounds represented by general Formula III for thetreatment of a medical condition or a combination of medical conditions,with is treatable by digoxin and a drug having an adverse interactionwith digoxin.

Prodrugs:

Prodrugs corresponding to the compounds presented herein arecontemplated in the context of the resent invention in order to provideactive agents that exhibit improved pharmacokinetic and/orpharmacodynamics profile, and/or a reduction in adverse effects. Asknown in the art, a prodrug is typically a chemical derivative of thecorresponding drug, being chemically modified such that a naturallyoccurring metabolic pathway in the subject's system can convert it tothe parent drug molecule during the time in which the prodrug/drug isstill present in the system. For instance, drugs that exhibit poorabsorption due to high hydrophobicity, can be modified to exhibitimproved bioavailability by introducing chemical functionalities thatincrease the solubility of the prodrug comparted to the parent activecompound. In other cases where the active compound exhibits adverseeffects in the GI-tract, the prodrug is a modified parent activecompound that is metabolized by enzymes back to the parent compoundsubstantially at the target tissues, organs or cells.

In the context of some embodiments of the present invention, theprodrugs are metabolized to afford the corresponding (parent) compoundby naturally occurring metabolic agents, such as enzymes. In someembodiments, the chemical group of the prodrug is bioliable orbiodegradable, such as in the case of an ester, which can be hydrolyzedby esterases to afford a hydroxyl of the parent compound.

According to some embodiments, the compounds presented herein exhibitseveral structural positions that can offer potential locations forchemical modifications on route to becoming suitable prodrugs. Forexample, a digoxin skeleton exhibits four hydroxyl groups which canpotentially be converted into, e.g., esters, under various conditions.Similarly, the digitoxin skeleton exhibits three hydroxyl moieties.Since each hydroxyl is located at a different chemical environment, isit chemically converted under varying conditions, thereby allowing theproduction of a single or a multiple hydroxyl-to-ester conversion. Thus,in the case of digoxin, a mono-, a bis-, a tris- or a tetra-modifiedparent compound can be afforded, offering a variety of prodrugs of thesame patent compound, each exhibiting a differentpharmacokinetic/pharmacodynamics profile.

In addition, R and R′ substituents (see, Formula I and Formula IIIrespectively) may exhibit a chemical position which is readily convertedinto another, bioliable (biodegradable) moiety, and thus can be used toafford prodrugs from the corresponding parent compound.

According to some embodiments, prodrugs of the compounds having thegeneral Formula I or general Formula III, are represented by generalFormula V:

wherein X is H or PD₃, andeach of PD₁-PD₄ is H or independently represents a modified hydroxylgroup turned into a bioliable functionality, provided that at least oneof PD₁-PD₄ is not H.

In some embodiments, the compounds presented herein are converted into aprodrug by modifying any one of the hydroxyl groups on the digoxin ordigitoxin moiety of the compounds into any one of a methoxymethyl ether,a tetrahydropyranyl ether, a t-butyl ether, an allyl ether, a benzylether, a t-butyldimethylsilyl ether, a t-butyldiphenylsilyl ether, anacetic acid ester (Ac), ethyl, propyl, butyl, t-butyl or pivalic acidester and a benzoic acid ester, in any combination. Each of theconverted hydroxyl functionalities may be metabolized (biodegraded) backto the parent hydroxyl by one or more metabolic and/or enzymaticsystems.

According to some embodiments, any one of PD₁-PD₄ in general Formula Vis selected from the group consisting of a methoxymethyl ether, atetrahydropyranyl ether, a t-butyl ether, an allyl ether, a benzylether, a t-butyldimethylsilyl ether, a t-butyldiphenylsilyl ether, anacetic acid ester (Ac), ethyl, propyl, butyl, t-butyl or pivalic acidester and a benzoic acid ester.

As can be seen in the Examples section that follows below, the exemplarydigoxin-derived compound DcB, according to some embodiments of thepresent invention, has been converted successfully into thecorresponding bit-acetyl (BisAcDcB) prodrug and the tris-acetyl(TrisAcDcB) prodrug (see, Example 6).

Isolation and Use of Na,K-ATPase Isoforms:

According to an aspect of some embodiments of the present invention,there is provided a method of determining an apparent affinity of thecompound represented by general Formula I to at least one isoform ofNa,K-ATPase, the method includes contacting an isoform of Na,K-ATPasewith the compound in an activity measurement setup and determining theapparent affinity of the compound to the isoform.

According to an aspect of some embodiments of the present invention,there is provided a method of isolating an isoform of Na,K-ATPase of amammal, the method includes:

transforming yeast cells with a clone that comprises an α chain sequenceand a β chain sequence of the Na,K-ATPase;

expressing the clone in the yeast cells; and

isolating the isoform,

wherein:

the α chain sequence is selected from the group consisting of α1, α2, α3and α4; and

the β chain sequence is selected from the group consisting of β1, β2 andβ3.

In some embodiments, the isolated isoform is α2β2.

In some embodiments, the isolated isoform is α2β3.

According to an aspect of some embodiments of the present invention,there is provided an isolated α2β2 isoform of Na,K-ATPase of a mammalhaving at least 70% purity.

According to an aspect of some embodiments of the present invention,there is provided an isolated α2β3 isoform of Na,K-ATPase of a mammalhaving at least 70% purity.

The yeast-expressed α/β subunits have distinct levels of glycosylationcompared to those in the human-expressed subunits. The distinctyeast-expressed glycosylation pattern does not substantially affect theactivity of the enzyme but may increase its stability.

According to an aspect of some embodiments of the present invention,there is provided an isolated α2β2 isoform of Na,K-ATPase of a mammalhaving a yeast-characterizing glycosylation pattern.

According to an aspect of some embodiments of the present invention,there is provided an isolated α2β3 isoform of Na,K-ATPase of a mammalhaving a yeast-characterizing glycosylation pattern.

In some embodiments, the isolated isoforms presented herein have anamino acid sequence which identical, substantially similar or derivedfrom any one of the isoform of human of Na,K-ATPase.

SEQ ID Nos. Description SEQ ID No. Amino acid sequence of α1 subunit ofhuman Na,K-ATPase (P05023) 1 Amino acid sequence of α2 subunit of humanNa,K-ATPase (P50993) 2 Amino acid sequence of α3 subunit of humanNa,K-ATPase (P13637) 3 Amino acid sequence of α4 subunit of humanNa,K-ATPase (Q13733) 4 Amino acid sequence of β1 subunit of humanNa,K-ATPase (P05026) 5 Amino acid sequence of HIS tagged β1 subunit ofhuman Na,K-ATPase (P05026) 6 Amino acid sequence of β2 subunit of humanNa,K-ATPase (P14415) 7 Amino acid sequence of HIS tagged β1 subunit ofhuman Na,K-ATPase (P14415) 8 Amino acid sequence of β3 subunit of humanNa,K-ATPase (P54709) 9 Amino acid sequence of HIS tagged β3 subunit ofhuman Na,K-ATPase (P54709) 10 Nucleotide sequence of α1 subunit of humanNa,K-ATPase (ATP1A1) 11 Nucleotide sequence of α2 subunit of humanNa,K-ATPase (ATP1A2) 12 Nucleotide sequence of α3 subunit of humanNa,K-ATPase (ATP1A3) 13 Nucleotide sequence of α4 subunit of humanNa,K-ATPase (ATP1A4) 14 Nucleotide sequence of HIS tagged β1 subunit ofhuman Na,K-ATPase (ATP1B1) 15 Nucleotide sequence of HIS tagged β2subunit of human Na,K-ATPase (ATP1B2) 16 Nucleotide sequence of HIStagged β3 subunit of human Na,K-ATPase (ATP1B3) 17 Nucleotide sequenceof HIS tagged human FXYD1 18 Amino acid sequence of HIS tagged humanFXYD1 19

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof. Throughout this application,various embodiments of this invention may be presented in a rangeformat. It should be understood that the description in range format ismerely for convenience and brevity and should not be construed as aninflexible limitation on the scope of the invention. Accordingly, thedescription of a range should be considered to have specificallydisclosed all the possible subranges as well as individual numericalvalues within that range. For example, description of a range such asfrom 1 to 6 should be considered to have specifically disclosedsubranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

It is expected that during the life of a patent maturing from thisapplication many relevant inhibitors exhibiting selectivity towardsα2-containing isoforms of Na,K-ATPase, as defined herein, will beuncovered and the scope of this term is intended to include all such newselective inhibitors a priori.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in anon-limiting fashion.

Example 1 Compound Synthesis

Compounds represented by general Formula I, according to embodiments ofthe present invention, were synthesized as a series ofperhydro-1-4-oxazepine derivatives of the third digitoxose of digoxin ordigitoxin following a general procedure described elsewhere [Adamczyk,M. et al., Steroids, 1995, 60(11), pp. 753-758] by selective oxidationwith NaIO₄ and reductive amination with NaCNBH₃. The compounds werepurified by HPLC and masses, ¹H-NMR and ¹³C-NMR spectra were determinedto verify both correctness of the structure and purity.

The structure and names based on of the amine substituents (R—NH₂), aswell as their theoretical and measured masses, are presented in Table 1below, which includes other digoxin derivatives with non-cyclic moieties(last eight compounds in Table 1).

TABLE 1 Theoretical Measured mass R Name mass (with Na+ ion) cyclopropylDcP 803.48 826.45 methylcyclopropane DMcP 817.50 840.44ethylcyclopropane DEcP 831.51 854.58 cyclobutyl DcB 817.50 840.43cyclopentyl DcPe 831.51 854.51 cyclohexyl DcHe 845.53 868.65 benzyl DBz853.50 876.44 methyl(3,3- DMDMcB 859.54 882.59 dimethylcyclobutane)methylcyclopropane DtxMcP 801.50 824.48 cyclobutyl DtxcB 801.50 824.44methyl DMe 777.47 800.57 ethyl DEt 791.48 814.52 2,2,2-trifluoroethylDCF₃ 845.45 868.14 propyl DP 805.50 828.27 iso-propyl DiP 805.50 828.41iso-butyl DiB 819.51 842.66 tert-butyl DtB 819.51 842.41methyl(trimethylsilyl) DTMS 849.51 872.77 (methylsulfonyl)methyl DMSM855.44 878.49 (methylsulfonyl)ethyl DESM 869.46 892.29(sulfonamide)ethyl DESA 870.45 893.47 azetidinyl DAz 818.49 841.55oxetanyl DOx 819.48 842.41 thietanyl DTh 835.45 858.37 histaminyl DHis857.50 880.45 (N,N-dimethylamine)ethyl DEDA 834.52 857.62 aminoethyl DED806.49 829.55 (methylsulfonyl)methyl DMSM 855.44 878.49

Names of compounds encompassed under Formula I wherein X=OH (digoxinderivatives) include Dcp, DMcP, DEcP, DcB, DcB, DcB, DcB and DMDMcB;names of compounds encompassed under Formula I wherein X=H (digitoxinderivatives) include, DtxMcP and DtxcB.

Example 2 Expression, Purification and Characterization of RecombinantHuman Na,K-ATPase Isolated Isoforms

Plasmid construction for the expression of α1β1, α2β1, α2β2 and α2β3Na,K-ATPase was conducted by generation of pHil-D2 expression vectorcontaining cDNA of human α₁ and His10 tagged human β₁ was describedpreviously [Lifshitz Y, et al., 2007, Biochemistry, 46(51), pp.14937-14950]. cDNA of human β₂ and β₃ in pSD5 were a gift from K.Geering, University Lausanne, Switzerland. Open reading frames andflanking regions of human β₂ and human β₃ were amplified by PCR usingprimers containing BglII and SalI cleavage sites. The resultingfragments were subcloned into pHil-D2-hα₂/His10-pβ₁ to createpHil-D2-hα₂/His10-hβ₂ and pHil-D2-hα₂/His10-hβ₃, respectively. Correctintegration and sequence was confirmed by sequencing.

Yeasts were grown in BMG (100 mM potassium phosphate pH 6, 1.34% yeastnitrogen base, 4×10⁻⁵% biotin, 0.3% glycerol) to OD 6-8 and expressionwas induced in BMM (100 mM potassium phosphate pH 6, 1.34% yeastnitrogen base, 4×10⁻⁵% biotin, 0.5% methanol added daily).

Pichia pastoris transformation, yeast growth, membrane preparation andHis-tag purification of recombinant human α1β1, α2β1, α2β2 and α2β3Na,K-ATPase were carried out essentially as described previously [KatzA, et al., 2010, J Biol Chem, 285(25), pp. 19582-19592; and Katz A, etal., 2014, J Biol Chem, 289(30), pp. 21153-21162].

Expression and purification of α1β1, α2β1, α2β2 and α2β3 complexes wasconducted in small scale whole cell lysates that were prepared asdescribe in Loayza, D. et al., Mol. Cell. Biol., 1998, 18, p. 779-789.Yeast membrane production and expression of recombinant Na,K-ATPase asdetergent soluble complexes was performed as described in Habeck, M. etal., J. Biol. Chem., 2015, 290, pp. 4829-4842, using modified lipidcontent (C12E8, 0.1 mg/ml; SOPS, 0.07 mg/ml; cholesterol, 0.01 mg/ml).

Na,K-ATPase α₁β₁, α₂β₁, α₂β₂ and α₂β₃ complexes were reconstituted withFXYD1 and purified in a mixture of 0.1 mg/ml C12E8, 0.07 mg/ml SOPS and0.01 mg/ml cholesterol.

The purified isoform complexes (0.3-0.5 mg/ml) were eluted from theBD-Talon beads in a solution containing Imidazole 170 mM, NaCl 100 mM;Tricine.HCl 20 mM pH 7.4; C12E8, 0.1 mg/ml; SOPS 0.07 mg/ml, cholesterol0.01 mg/ml, glycerol 25%, by gravity-column. The proteins were stored at−80° C. Protein purity was determined by gel electrophoresis and proteinconcentration was determined with BCA (B9643 Sigma).

The specific Na,K-ATPase activity was highest for α₁β₁ (16.4±0.7μmol/mg/min) followed by α₂β₁ (10.9±0.6) and α₂β₃ (10.7±1.9). The α₂β₂isoform had the lowest activity (8.4±1.4). The second significantlydifferent parameter is the apparent K⁺-affinity. K_(0.5) K⁺ for α₂β₁ was2.7±0.14 mM compared to 1.47±0.06 mM for α₁β₁. α₂β₂ and α₂β₃ had an evenlower affinity than α₂β₁ with apparent K_(0.5) values of 7.4±0.19 mM and6.4±0.50 mM, respectively. Na-titrations revealed that the affinity forNa⁺-ions was not different between α₁β₁ and α₂β₁ whereas α₂β₂ and α₂β₃had a somewhat higher Na-affinity.

The reduced apparent affinity for K⁺ together with an increased affinityfor Na⁺ indicates that the conformational equilibrium of α₂β₂ and α₂β₃might be shifted towards E1. In order to test this hypothesis, theapparent affinity of vanadate was determined for all four isoformcomplexes. Vanadate is a phosphate analogue that binds to the E2conformation, mimicking the transition state E2PK₂ duringdephosphorylation, thus inhibiting the enzyme. All three α2 isoforms hada lower vanadate affinity compared to α₁β₁ (0.48 μM). α₂β₂ had thelowest vanadate affinity (34 μM) followed by α₂β₃ (19 μM) and α₂β₁ (3.5μM). Thus, the order of inhibition by vanadate equals the order ofpotassium activation (K_(0.5)K⁺ and K_(i) vanadate α₁β₁<α₂β₁<α₂β₃<α₂β₃)supporting the hypothesis proposed above.

Example 3 Selective Inhibition Assays of Isolated Na,K-ATPase

To screen for isoform selectivity of the digoxin derivatives we comparedinhibition of Na,K-ATPase activity of purified detergent-soluble humanisoform complexes α1β1FXYD1, α2β1FXYD1, α2β2FXYD1 and α2β3FXYD1.Although all the preparations and assays were conducted with FXYD1 inorder to stabilize the complexes, the FXYD1 suffix is omitted in namingof isoform complexes for simplicity.

Na,K-ATPase activity of α/βFXYD1 complexes was measured over one hour at37° C. in a medium containing 130 mM NaCl, 5 mM KCl, 3 mM MgCl₂, 1 mMEGTA, 25 mM Histidine, pH 7.4 and 1 mM ATP using the PiColor Lock goldmalachite green assay (Inova Biosciences).

The Na,K-ATPase activities were α1β1, 21.5±5.3 moles/min/mg; α2β1,18.7±1.8 moles/min/mg, and α2β3, 10.7±1.9 moles/min/mg protein. Asdiscussed below, an important kinetic property in relation to inhibitionby cardiac glycosides is K_(0.5) for activation by K: α1β1—1.25±0.05 mM,α2β1—2.7±0.14 mM and α2β3 6.4±0.50 mM, respectively.

Selectivity of the compounds for various isolated isoforms of humanNa,K-ATPase was determined essentially as described before [Katz, A. etal., J Biol Chem., 2010, 285(25), pp. 19582-19592].

ATPase activity assays as well as titrations with NaCl, KCl and vanadatewere performed as described in Lifshitz-2007 and Loayza-1998 usingPiColorLock™ malachite green assay (Inova Bioscience). Inhibitor assayswere performed as described in Katz-2010. [³H]ouabain binding andK⁺-[³H]digoxin displacement assays were performed as described inKatz-2010.

The percent inhibition VCG/V0 was calculated and Ki values were obtainedby fitting the data to the function VCG/V0=Ki/([CG]+Ki)+c (CG stands forcardiac glycoside). Inhibition was estimated in 3-5 separate experimentsand average Ki values±standard error of the mean (SEM) were calculated.The ratios Ki α1β1/α2β1, α1β1/α2β2 and α1β1/α2β3 was calculated for eachcompound.

Table 2 shows the Ki values and selectivity ratios (Ki α1β1:α2β1, Kiα1β1:α2β2 and α1β1:α2β3) for inhibition of Na,K-ATPase activity ofcompounds according to embodiments of the present invention, as well assome digoxin derivatives having a non-cyclic moiety, compared to digoxinand digitoxin. Table 2 is arbitrarily sorted according to column “Kiratio α1β1/α2β3” marked by “*”

TABLE 2 No. of C Selectivity atoms in Ki in nM ± SEM Ki ratio Ki ratio*Ki ratio Compound Name R α1β1 α2β1 α2β2 α2β3 α1β1/α2β1 α1β1/α2β2α1β1/α2β3 DcB (cyclic) 4 135 ± 11    8 ± 1.25  6 ± 1    4 ± 0.15 16.922.2 33.6 DMcP (cyclic) 4  95.8 ± 13.7 18.3 ± 1.6  8.0 ± 0.8  4.3 ± 0.65.2 12 22.2 DESM (sulfonyl) 2 464 ± 14 49.2 ± 1.9 31.7 ± 3.2 24.7 ± 2.19.4 14.6 18.8 DiB 4   92 ± 8.9 20.6 ± 1.4   10 ± 0.8  5.8 ± 0.6 4.4 9.016 DESA (sulfonamide) 2 301 ± 23 38.9 ± 2.2 31.5 ± 4.4 20.1 ± 0.9 7.79.5 15 DiP 3   149 ± 20.7 28.9 ± 1.7 16.7 ± 1.9 10.3 ± 1.8 5.1 8.9 14.4DcP (cyclic) 3  109 ± 6.2  14.6 ± 11.6 13.0 ± 1.3  8.1 ± 1.36 7.5 8.513.4 DEcP (cyclic) 5 86.1 ± 7   14.3 ± 2   12.1 ± 1.9 7.2 ± 1  6.02 7.112.02 DMSM (sulfonyl) 3  944 ± 123  137 ± 9.8  123 ± 7.3   89 ± 8.7 6.97.7 10.6 DBz (cyclic) 6  57.9 ± 15.5 10.1 ± 2.2  6.8 ± 1.2  5.6 ± 1.65.7 8.5 10.3 DcH (cyclic) 6 70.4 ± 4.1 15.2 ± 3.7 15.3 ± 2.9 11.7 ± 4.54.6 4.6 10.1 DCF₃ 2   119 ± 15.0 28.6 ± 0.9 18.1 ± 1.9 12.4 ± 1.5 4.16.5 9.6 DEt 2 137.9 ± 12.6 23.2 ± 0.9 16.4 ± 1.6  14.4 ± 1.27 5.9 8.39.5 DMe 1  103 ± 5.6 15.3 ± 1.2 20.36 ± 1.8  10.8 ± 0.6 6.7 5.1 9.5 DP 387.7 ± 7.9  18.3 ± 1.68 10.5 ± 1.8  9.8 ± 1.1 4.8 8.3 8.8 DtB 4   135 ±12.1 21.6 ± 5.6 18.4 ± 1.1  16.3 ± 0.28 6.2 7.3 8.2 DMDMcB (cyclic) 731.6 ± 0.5  8.6 ± 1.4  5.1 ± 0.5  3.9 ± 0.7 3.69 6.16 8.19 DtxcB(cyclic) 4 30.7 ± 7.2  5.4 ± 0.5  5.3 ± 0.8  4.3 ± 0.6 5.6 5.8 7.1DtxMcP (cyclic) 4 25 ± 4  4.2 ± 0.4  5.4 ± 1.2  3.7 ± 0.5 5.9 4.6 6.7DTMS (sily1) 4  72.6 ± 17.6 24.3 ± 4.0 14.3 ± 1.3 11.1 ± 1.6 3 5.1 6.5Digoxin 0   268 ± 13.8 58.7 ± 5.4   58 ± 1.9 42.8 ± 3.0 4.5 4.6 6.2 DcPe(cyclic) 5 138 ± 21 33.4 ± 7.5  33.5 ± 11.9 27.6 ± 9.5 4.1 4.1 5Digitoxin 0   89 ± 15.8 29.5 ± 2.7 40.7 ± 6.7 28.8 ± 5.9 3 2.1 3.1

As can be seen in Table 2, some compounds show an even greaterselectivity ratio towards α2β2 and α2β3, particularly α2β3. Based on theratios of Ki values, several derivatives show significantly improvedselectivity for α2β3 compared to α2β1 over α1β1, in particular DMcP andDcB. DMcP and DcB show exceptional selectivity for α2β2 and α2β3 overα1β3 of 22-fold and 33-fold, respectively, and very low Ki values forinhibition of α2β3 (Ki about 4 nM). The full inhibition curves of DMcPand DcB emphasize the extent of the difference between α1β1 and α2β3.

Bearing in mind the large differences in K_(0.5) potassium ions, a pointto be aware of in analyzing the results presented in Table 2 is thewell-known K-cardiac glycoside antagonism. Digoxin itself has moderateselectivity for α2β1 over α1β1 (about 4-fold) and the data in Table 2shows increased selectivity for α2β3 over α1β1 (about 6-fold). Theα2β1-selectivity of digoxin is attributed to a combination of increasedbinding affinity for α2 over α1 and also reduced K-digoxin antagonism inthe Na,K-ATPase reaction conditions (with K, 5 mM). Similarly, theincreased selectivity of digoxin for α2β3 compared to α2β1 isattributable to reduced K-cardiac glycoside antagonism due to the higherK_(0.5) K of α2β3 compared to α2β1.

The compounds presented herein show a notable increase of the ratio ofKi's α1β1:α2β3 compared to α1β1:α2β1, which must be partly due to thereduced K-cardiac glycoside antagonism. However, the difference for themost α2β3-selective compounds is significantly greater than that fordigoxin and cannot be explained only by this factor. In particular,there is a distinct structural effect in that the maximalα2β3-selectivity is seen for R-substituents with cyclic moiety, as wellas those with four carbon atoms. In the case of DMcP and DcB, the Ki forα1β1 is about 2-3-fold lower than for digoxin itself but the Ki for α2β3is about 10-fold lower than for digoxin, thus raising selectivity forα2β3 over α1β1 to more than 22-fold and more than 33-fold, respectively.It is also noticeable that for derivatives with five or a higher numberof carbon atoms in the R-substituents, such as DEcP, DcPe, DcH, DBz andDDMcB, although the Ki values for all isoforms are all lower than fordigoxin itself, the selectivity for α2β3 over α1β1, 10-12-fold, is alsolower than the selectivity observed in four carbon R-substituents, DMcPand DcB. The same is true for compounds with R-substituent three carbonsor shorter.

Another structural insight comes from the results obtained for twodigitoxin derivatives (DtxMcP and DtxcB) compared with the digoxinderivatives DMcP and DcB. As seen in Table 2, the Ki values arerelatively low for all three isoform complexes α1β1, α2β1 and α2β3,being reduced about 10-fold compared to digoxin. Consequently, thesedigitoxin derivatives showed little increase in selectivity for α2β3compared to digoxin itself. Thus, the absence of a single OH group inposition 12 of the steroid moiety of digitoxin reduced the effect of thesugar modification on α2β3-selectivity.

Thus, modification of the third digitoxose moiety of digoxin, but not ofdigitoxin, with cyclic substituents such as McP and cB confers notableα2β3-selectivity, presumably due to selective interaction with β3.

Example 4 Inhibition of Na,K-ATPase Activity in Permeabilized Bovine NPECells

In order to prove the concept of selective inhibition of the compoundspresented herein in Na,K-pumps of native ciliary epithelium, inhibitionassays using Na,K-ATPase in NPE cells isolated from the ciliary bodydissected out of bovine eyes were conducted.

Isolation of ciliary epithelium PE and NPE cells was carried out aspreviously described [Edelman, J. L. et al., 1994, Am J Physiol, 266(5Pt 1), pp. C₁₂₁₀-1221], with some modifications, using 15-20 freshbovine eyes. Ciliary bodies were isolated from bovine eyes and washed inringer solution. The tissue was treated with trypsin and homogenizedfollowed by separation on a density gradient of Metrizamide, whichseparates between the NPE and PE cells.

Using isolated human isoforms to calibrate the response of theantibodies, NPE cell lysates were shown to contain about 70% α2 and 30%α1, while PE were shown to contain about 90% α1 and 5-10% α2. Afterunmasking the Na,K-ATPase by treating the cells with alamethicin,Na,K-ATPase activity was measured and was found to be 0.195±0.027 and0.035±0.008 nmoles/mg protein/min in NPE and PE cells, respectively.Ouabain-sensitive fractions of total ATPase activity were about 65% and35% for NPE and PE cells, respectively.

To determine Na,K-ATPase activity in the cells, the cells were incubatedwith 0.8 mg/ml alamecithin for 30 minutes at room temperature prior totransfer to the reaction medium containing 130 mM NaCl, 5 mM KCl, 3 mMMgCl₂, 25 mM histidine, pH 7.4, 1 mM EGTA, 1 mM sodium azide, 0.5 mMATP, and were then incubated for 45 minutes at 37° C., with or withoutthe tested inhibitors as indicated, or 0.5 mM ouabain to determine theouabain insensitive ATPase activity. The data was fitted to one or twosites inhibition model.

Table 3 presents Ki values for inhibition of NPE Na,K-ATPase activity bydigoxin, DMe, DMcP and DcB, fitted to a single site inhibition model.

TABLE 3 Compound Ki, nM ± SEM n 1 site model Digoxin 91.7 ± 10.2 4 DMe15.6 ± 1.3  4 DMcP 7.9 ± 2.2 5 DcB 17.3 ± 2.5  4 2 sites model DcB Ki α26.9 ± 2 Ki α1 151 ± 7.6 4 (A α2 0.66 ± 0.090 A α1 0.34 ± 0.097)

As can be seen in Table 3, the Ki values of the derivatives are alllower than that of digoxin. Since NPE cells contain about 70% α2 and 30%α1, Na,K-ATPase activity and inhibition should reflect the properties ofthe isoform mixture. Indeed, the detailed inhibition curve for the mostα2β3-selective compound, DcB, was fitted better by a two site model,compared to fitting according to a one site model.

As can be seen in Table 3, the two site model provides the best fitparameters of 66% α2, Ki 6.9±2 nM; 34% α1, Ki 151±7.6 nM (Ki α1/α2=22),which are quite close to the proportions of α2:α1 estimated in theimmunoassays, and the selectivity ratio Ki α1β1/α2β3 is about 33.

Thus, it can be concluded that the selectivity properties of the digoxinderivative DcB observed with purified human isoforms is corroborated bythe results obtained using intact NPE cells.

Example 5 Reduction of Intraocular Pressure

These experiments examined the effects of topically administeredα2β3-selective digoxin derivatives, according to some embodiments of thepresent invention, DiB, DMcP and DcB, on IOP in rabbits. Due to thelower Ki for inhibition of α2β3, compared to digoxin, and the highhydrophobicity, these compounds were predicted to both permeate thecornea well and efficiently inhibit the α2β3 in the NPE ciliaryepithelium, thus reducing inflow of aqueous humour and IOP.

New Zealand white rabbits (3-3.5 kg) about 1 year old, of either sex,were housed in pairs in cage in animal room conditions on a reversed,12-hour dark/light cycle. For the experiments the animals weretransferred to rabbit restrainers in a quiet and calm atmosphere. Noocular abnormalities were detected prior or during the experiments.

IOP measurements were made with a pneumatonometer (Model 30, Reicherttechnologies) either after raising IOP with 4-aminopyridine (4AP; 1 drop40 mg/ml), or on basal IOP after addition of one drop of 1 mM solutionof digoxin derivatives to the right eye (RE) and one drop of PBS to theleft eye (LE) that served as control.

For comparison of effects of digoxin derivatives, such as DcB with aknown glaucoma drug Latanoprost, three groups of five rabbits were used.Rabbits treated with Latanoprost, received the medication every day for5 days before the start of the experiment. On the day of the experimentrabbits were treated at 5 minutes interval with one drop of 1 mM DcB,one drop of 0.005% Latanoprost (Xalatan™, Pfizer) or one drop each ofDcB and Latanoprost (RE), or normal saline (LE, Control). IOP wasmeasured every hour for 12 hours (DcB and Latanoprost alone) and after24 hours (DcB with Latanoprost). Basal IOPs in both eyes, without anymedication, were measured 5 days before and on the day of theexperiment. All eyes were examined routinely by ophthalmic examinationsand were free of any abnormalities. Corneal thickness (μm) was measuredusing an ultrasonic pachometer (Sonogage pachometer, Cleveland, USA).

Stock solutions of the tested compounds were dissolved in ethanol, andfreshly diluted in phosphate buffer (PBS) for each experiment, such thatthe final ethanol concentration did not exceed 1%.

The first set of experiments examined the effects of the α2-inhibitorcompounds, according to embodiments of the present invention, whenapplied just before 4AP, used as a pharmacological tool to transientlyraise IOP. One drop of the tested compound (0.01-0.3 mM) was appliedtopically to the rabbit's eyes prior to the 4AP, and IOP was thenmeasured over 5 hours.

FIGS. 1A-D present comparative plots of IOP as a function of time,showing the dose response of α2-inhibitor compounds, according to someembodiments of the present invention, in lowering IOP in live rabbits,wherein FIG. 1A shows the results obtained for DiB, FIG. 1B shows theduration of the effect of DiB while 4AP is added every 2 hours so as tomaintain the raised control IOP, FIG. 1C shows the results obtained forDMcP, and FIG. 1D shows the results obtained for DcB.

As can be seen in FIG. 1A, DiB, having Ki α1β1:α2β3 ratio of 16-fold)prevents the rise of the 4AP-induced IOP at concentrations of more than0.030 mM while at 10 μM it is still effective. As can be seen in FIG.1B, the duration of the DiB effect was shows that 1 mM DiB is effectivefor about 8 hours before the IOP begins to rise back up to controllevels, which is a significantly long effect.

As can be seen in FIGS. 1B and 1C, DMcP, having a Ki α1β1:α2β3 ratio of22-fold, and DcB, having a Ki α1β1:α2β3 ratio of 33-fold, exhibitsimilar IOP reduction as DiB (FIG. 1A), but in even lowerconcentrations. As can be seen in FIGS. 1B and 1C, low concentrations of0.01 mM of DMcP or DcB are sufficient to prevent the 4AP-induced rise inIOP.

As can further be seen in FIGS. 1B and 1C, at higher concentrations of0.1-0.3 mM, the IOP is reduced to levels which are significantly lowerthan the starting IOP. The latter observation implies that thesecompounds could reduce basal IOP even in the absence of 4AP.

FIGS. 2A-D present comparative plots of IOP as a function of time,demonstrating the capacity of the α2-inhibitor compounds, according tosome embodiments of the present invention, to lower IOP below basallevels compared to a buffer control when administered topically to oneeye of a rabbit, while the other eye received PBS as a control, whereinFIG. 2A shows the lack of effect of digoxin, FIG. 2B shows the lack ofeffect of DiB (a non-cyclic moiety inhibitor), FIG. 2C shows the notableof effect of DMcP, and FIG. 2D shows the notable of effect of DcB.

As can be seen in FIG. 2A-B, neither digoxin nor DiB had a significanteffect on lowering the basal IOP. This observation coincides with otherobservations with digoxin or other non-cyclic moiety digoxinderivatives, DMe, DGlyN, the latter two exhibiting enhanced selectivityfor α2β1.

As can be seen in FIGS. 2C-D, both DMcP and DcB significantly reducedthe basal IOP by 20-25% (about 4 mm Hg for rabbit with a basal IOP of 17mm Hg) over the test period of 4-5 hours. A higher concentration of DcB(2 mM) reduces the IOP similarly to 1 mM DcB.

This observation indicates that the α2-inhibitor compounds, according tosome embodiments of the present invention, can be used effectively totreat medical conditions where there in a need to lower IOP below whatis co considered to be a normal pressure, such as cases of low-tensionglaucoma and normal-tension glaucoma.

A final set of experiments compared the effectiveness and duration ofthe effects of topical administration of DcB on basal IOP, with those ofa widely used anti-glaucoma drug, Latanoprost, applied either alone orin combination (co-administration).

Groups of 5 rabbits were treated once a day for 5 days with Latanoprostand on the sixth day with DcB, Latanoprost, or DcB/Latanoprostcombination. IOP measurements were made for the next 12 hours or over 24hours for the group treated with both DcB and Latanoprost.

FIGS. 3A-C present comparative plots of IOP as a function of time,demonstrating the effect of α2-inhibitor compounds, according to someembodiments of the present invention, to potentiate the drug Latanoprostin lowering IOP below basal levels, wherein FIG. 3A shows the effect ofDcB alone, FIG. 3B shows the effect of Latanoprost alone, and FIG. 3Cshows the effect of co-administering DcB with Latanoprost.

As can be seen in FIGS. 3A-C, compared to the basal IOP values of 17-18mmHg, after 3-8 hours the steady-state IOP was lower by 3.5±0.15,2.6±0.11 and 3.44±0.39 mmHg with DcB, Latanoprost and DcB/Latanoprost,respectively, corresponding to steady-state IOP's of 75-80%, 85% and75-80% of the unchanged control values.

As can further be seen in FIGS. 3A-C, DcB, an α2-inhibitor compoundaccording to some embodiments of the present invention, was about 25%more effective than Latanoprost in reducing the basal (normal) levels ofIOP.

The combination of DcB and Latanoprost was similar in effect to DcBalone; however, with respect to the duration of the effect, for eitherDcB and Latanoprost, applied alone, the IOP returned to the control(normal) value after 12 hours, but with the combined DcB/Latanoprosttreatment the low IOP was maintained for a significantly longer periodand it returned to the control value only after 24 hours.

Latanoprost was applied for 5 days prior to the day of measurement, dueto reports that this pre-treatment produces optimal effects on IOP inhumans, although in these experiments with rabbits it seems that thiswas unnecessary since the effects of Latanoprost were observed acutelyand had dissipated completely after 24 hours.

The animals which received the treatment presented in FIGS. 3A-C wereused for corneal thickness measurements after topically administeringdrops of DcB or Latanoprost to 4 rabbits once a day for 5 additionaldays, totaling 6 days of treatment with DcB or Latanoprost, and theresults are presented in Table 4 below.

TABLE 4 Treatment to Pachymetry (mm) Rabbit RE RE LE 1 DcB 455 456 2 DcB480 456 3 DcB 400 396 4 DcB 382 374 5 Latanoprost 467 471 6 Latanoprost489 498

As seen in Table 4, there was no detectable effect of either drug oncorneal thickness. In addition, by inspection, no significant redness orocular irritation was observed.

In order to assess whether topical application of DcB damaged thetissues of the eye including cornea, iris, lens, cilary body, retina,choroid and sclera, a histological examination was conducted aftertopical application, using one drop of 1 mM DcB daily for one week inone eye of each animal, with the other eye treated topically with onedrop of PBS and serving as the control. Animals were sacrificed, eyeswere removed, fixed in 10% neutral buffered formalin, processedroutinely for histological examination, trimmed at 4 μm, and stainedwith hematoxylin and eosin. No significant histological differences wereobserved in a comparative analysis of the treated and untreated eyes,indicating that topical treatment with DcB for a week does not causetissue damage in the eye.

Example 6 Prodrug Synthesis and Characterization

Prodrugs of the compounds presented herein were developed in order toimprove the pharmacokinetic profile of the active compounds, e.g., toavoid potential toxic effects in the cornea. The synthetic strategyincluded preparation of substantially inactive derivatives of thecompounds, which are ineffective as an inhibitor of the major isoform ofthe Na,K-ATPase in the cornea (mainly the α1β1 isoform), and which canpenetrate into the eye and thereafter are converted into the active formby biodegradation processes. The biodegraded and active form of theprodrug can then inhibits the α2β2/3 complex in the ciliary NPE cellsand reduces inflow of aqueous humor.

Synthesis:

The synthesis of an exemplary prodrug compound, according to someembodiments of the present invention, 3′,3″-bisacetyl digoxincyclobutane (bisAcDcB), from an exemplary compound according to someembodiments of the present invention, DcB, is illustrated in Scheme 2below.

In this example, the bisacetyl derivative (IUPAC name(2R,3R,4S,6R)-3-(((2S,4S,5R,6R)-4-acetoxy-5-((4-cyclobutyl-2-methyl-1,4-oxazepan-7-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-6-(((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12,14-dihydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-2-methyltetrahydro-2H-pyran-4-ylacetate) was afforded by stirring for 6 hours at room temperature asolution of DcB (10 mg, 12.3 μmol, MW=817) and 4-dimethylaminopyridine(DMAP) catalyst (0.3 mg, 2.4 μmol, MW=122) in 2 ml toluene, 2 mlpyridine and 0.7 ml acetic anhydride.

Thereafter, the resulting mixture, rich in the bis-acetylatedderivative, was extracted with water and dichloromethane, and subjectedto final wash with HCl-acidified water pH of about 4. The resultingresidue was dried over MgSO₄, and purified in normal phase HPLC using asilica gel column.

The synthesis of an exemplary prodrug compound, according to someembodiments of the present invention, 12,3′,3″-trisacetyl digoxincyclobutane (trisAcDcB), from an exemplary compound according to someembodiments of the present invention, DcB, is illustrated in Scheme 3below.

In this example, the trisacetyl derivative (IUPAC name(2R,3R,4S,6R)-6-(((3S,5R,8R,9S,10S,12R,13S,14S,17R)-12-acetoxy-14-hydroxy-10,13-dimethyl-17-(5-oxo-2,5-dihydrofuran-3-yl)hexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)-3-(((2S,4S,5R,6R)-4-acetoxy-5-((4-cyclobutyl-2-methyl-1,4-oxazepan-7-yl)oxy)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-2-methyltetrahydro-2H-pyran-4-ylacetate) was afforded by stirring for 24 hours at room temperature asolution of DcB (10 mg, 12.3 μmol, MW=817) and 4-dimethylaminopyridine(DMAP) catalyst (0.3 mg, 2.4 μmol, MW=122) in 2 ml toluene, 2 mlpyridine and 0.7 ml acetic anhydride.

Thereafter, the resulting mixture, rich in the tris-acetylatedderivative, was extracted with water and dichloromethane, and subjectedto final wash with HCl-acidified water pH of about 4. The resultingresidue was dried over MgSO₄, and purified in normal phase HPLC using asilica gel column.

Calculated octanol-water partition coefficient (C log P) for DcB is3.43, for a mono-acetyl derivative of DcB is 3.93, for the bisAcDcBderivative is 4.37, and for the trisAcDcB derivative is 4.85 (C Log Psoftware ALOGPS 2.1 [Tetko, I. V. et al., J. Chem. Inf. Comput. Sci.,2002, 42, 1136-45])

In Vitro Inhibition Activity:

The results of the inhibition assay of purified human Na,K-ATPase(α1β1FXYD1 and α2β1FXYD1) by DcB, bisAcDcB and trisAcDcB are presentedas Ki values in Table 5 below.

TABLE 5 Ki of α1β1FXYD1 Ki of α2β1FXYD1 Compound [nM] [nM] DcB 135 ± 11   8 ± 1.25 BisAcDcB 1180 ± 280 395 ± 50 TrisAcDcB 13200 ± 6500 2500 ±490

As can be seen in Table 5, the Ki values of α1β1 by the exemplarybisAcDcB prodrug derivative and the exemplary trisAcDcB prodrugderivative are about 9-fold and 100-fold higher than that observed forthe corresponding exemplary compound DcB, respectively, while the Kivalues of α2β1 by bisAcDcB and trisAcDcB are about 50-fold and 300-foldhigher than that observed for the corresponding DcB, respectively.

In Vivo Activity—Lowering of Basal IOP:

One 30 μl drop of trisAcDcB or vehicle was applied to the right or lefteye of two rabbits, respectively, at 0.05, 0.1, and 0.2 mMconcentrations. The IOP was then measured every 1-2 hours over 12 hours.

FIG. 4 presents comparative plots of IOP as a function of time,demonstrating the capacity of the trisAcDcB prodrug of the α2-inhibitorcompound DcB, according to some embodiments of the present invention, tolower IOP below basal levels compared to a buffer control whenadministered topically to one eye of a rabbit, while the other eyereceived PBS as a control.

As can be seen in FIG. 4, no effect was observed at the first fourhours, and after that the IOP in the drug-treated samples dropped from15-16 mmHg to a minimum of 12 mmHg within 5-6 hours, before returning tothe control level after 12 hours. In addition, the maximal effect of thetrisAcDcB on IOP was essentially equal to that produced by the parentDcB (see, for example, FIG. 2D). The comparison with the parent DcBcompound shows that significantly lower doses of the prodrug arerequired to produce the maximal effect.

Thus, it can be concluded that acetyl prodrugs of the compoundspresented herein exhibit low affinity and weak inhibition of α1β1isoform, which is the major isoform in the cornea, making it unlikelythat topical application of the prodrug in human eye would cause cornealswelling or other toxic adverse effects. The lag of four hours prior toa detectable effect of the prodrug suggests that this time is requiredfor the intra-ocular esterases to hydrolyze the acetyl groups andregenerate DcB within the anterior chamber of the eye. Moreover,significantly lower doses of prodrug are required to achieve acomparable effect of the parent compound. This finding is consistentwith a higher permeability through the cornea compared to DcB (C log P3.43) due to a higher lipophilicity of the esters (triAcDcB C log P4.85).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting. In addition, any priority document(s) of this applicationis/are hereby incorporated herein by reference in its/their entirety.

What is claimed is:
 1. A pharmaceutical composition comprising as activeingredients: at least one ingredient selected from the group consistingof a prostaglandin analog, a β-blocker, an adrenergic agent, anα2-adrenergic receptor agonist, a miotic agent, a carbonic anhydraseinhibitor and a cholinergic agonist; and a compound represented byFormula III:

including any pharmaceutically acceptable salt, hydrate, solvate,enantiomer and diastereomer thereof, and any mixtures thereof, and apharmaceutically acceptable carrier, wherein: X is H or OH; R′ isselected from the group consisting of OH, C₁-C₆ alkyl, C₁-C₆ haloalkyl(C₁-C₆ alkyl substituted with at least one halo),—(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂,—(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂and —(CR^(b)R^(c))n-NH₂; Y is O or S; R1, R2 and R3 are eachindependently H or a C₁-C₄ alkyl; Ra is a C₁-C₄ alkyl; Rb, Rc and Rd areeach independently selected from H, a C₁-C₄ alkyl and a C₁-C₄hydroxyalkyl; Re is selected from a C₁-C₄ alkyl and a C₁-C₄hydroxyalkyl; and n is 0, 1 or
 2. 2. The pharmaceutical composition ofclaim 1, being packaged in a packaging material and identified in print,or on said packaging material, for use in reducing intraocular pressure(IOP).
 3. The pharmaceutical composition of claim 1, wherein said atleast one ingredient is said prostaglandin analog.
 4. The pharmaceuticalcomposition of claim 1, wherein said at least one ingredient is saidβ-blocker.
 5. The pharmaceutical composition of claim 1, wherein said atleast one ingredient is said adrenergic agent.
 6. The pharmaceuticalcomposition of claim 1, wherein said at least one ingredient is saidα2-adrenergic receptor agonist.
 7. The pharmaceutical composition ofclaim 1, wherein said at least one ingredient is said miotic agent. 8.The pharmaceutical composition of claim 1, wherein said at least oneingredient is said carbonic anhydrase inhibitor.
 9. The pharmaceuticalcomposition of claim 1, wherein said at least one ingredient is saidcholinergic agonist.
 10. A method of treating a heart condition in asubject in need thereof, comprising co-administering to the subject atherapeutically effective amount of: an agent selected from the groupconsisting of a β-blocker, an anticoagulation agent, anangiotensin-converting-enzyme inhibitor and an angiotensin II receptorantagonist; and a compound represented by Formula III:

including any pharmaceutically acceptable salt, hydrate, solvate,enantiomer and diastereomer thereof, and any mixtures thereof, wherein:X is H or OH; R′ is selected from the group consisting of OH, C₁-C₆alkyl, C₁-C₆ haloalkyl (C₁-C₆ alkyl substituted with at least one halo),—(CR^(b)R^(c))nSi(R^(a))₃, —(CR^(b)R^(c))n-C(═Y)—NR₁R₂,—(CR^(b)R^(c))n-C(═Y)—NHOH, —(CR^(d)R^(e))n-C(═Y)—COOR₃, —NHC(═Y)NR₁R₂and —(CR^(b)R^(c))n—NH₂; Y is O or S; R₁, R₂ and R₃ are eachindependently H or a C₁-C₄ alkyl; Ra is a C₁-C₄ alkyl; Rb, Rc and Rd areeach independently selected from H, a C₁-C₄ alkyl and a C₁-C₄hydroxyalkyl; Re is selected from a C₁-C₄ alkyl and a C₁-C₄hydroxyalkyl; and n is 0, 1 or
 2. 11. The method of claim 10, whereinsaid agent is said β-blocker.
 12. The method of claim 10, wherein saidagent is said anticoagulation agent.
 13. The method of claim 10, whereinsaid agent is said angiotensin-converting-enzyme inhibitor.
 14. Themethod of claim 10, wherein said agent is said angiotensin II receptorantagonist.